Download Listeria monocytogenes in fish farming and processing

Department of Food and Environmental Hygiene
Faculty of Veterinary Medicine
University of Helsinki, Finland
LISTERIA MONOCYTOGENES IN FISH
FARMING AND PROCESSING
Hanna Miettinen
ACADEMIC DISSERTATION
To be presented with the permission of the Faculty of Veterinary Medicine,
University of Helsinki, for public examination in Auditorium P3, Porthania,
Yliopistonkatu 3, on November 11th, 2006 at 10 o’clock a.m.
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Supervising Professor
Professor Hannu Korkeala, DVM, PhD
Faculty of Veterinary Medicine
Department of Food and Environmental Hygiene
University of Helsinki, Finland
Supervisors
Professor Hannu Korkeala, DVM, PhD
Faculty of Veterinary Medicine
Department of Food and Environmental Hygiene
University of Helsinki, Finland
Docent Gun Wirtanen, D.Sc. (Tech)
Bioprocessing
VTT Technical Research Centre of Finland
Espoo, Finland
Technology Manager Laura Raaska, PhD
Bioprocessing
VTT Technical Research Centre of Finland
Espoo, Finland
Rewiers
Professor Weihuan Fang, MS, PhD
Institute of Preventive Veterinary Medicine
Zhejiang University
Hangzhou, China
Professor Marcello Trevisani, DVM, Dipl. ECVPH
Food Hygine and Technology
Faculty of Veterinary Medicine
University of Bologna, Italy
Opponent
Docent Riitta Maijala, DVM, PhD, Dipl. ECVPH
Animal Health and Welfare Unit
Department of Food and Veterinary Control
Finnish Food Safety Authority, Finland
ISBN 952-92-1078-7 (Paperback)
ISBN 952-10-3440-8 (PDF)
Helsinki University Printing House
Helsinki 2006
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CONTENTS
ACKNOWLEDGEMENTS.......................................................................................................VI
ABBREVATIONS.................................................................................................................... VII
ABSTRACT .................................................................................................................................. 1
LIST OF ORIGINAL PUBLICATIONS ................................................................................... 3
1 INTRODUCTION..................................................................................................................... 4
2 REVIEW OF THE LITERATURE......................................................................................... 5
2.1 Listeria monocytogenes.......................................................................................................................5
2.1.1 Listeria monocytogenes............................................................................................................ 5
2.1.2 Listeriosis ................................................................................................................................. 5
2.1.3 Isolation and identification....................................................................................................... 7
2.1.4 Subtyping ................................................................................................................................. 9
2.2 Growth characteristics of Listeria monocytogenes..........................................................................9
2.3 Thermal resistance of Listeria monocytogenes ..............................................................................12
2.4 Listeria monocytogenes in nature, seafood industry and seafood products................................15
2.4.1 Occurrence of Listeria monocytogenes in environment......................................................... 15
2.4.2 Sources and routes of Listeria monocytogenes contamination and its occurrence in
seafood industry ..................................................................................................................... 16
2.4.3 Occurrence of Listeria monocytogenes in seafood products.................................................. 22
2.5 Control of Listeria monocytogenes in fish processing ...................................................................26
3 AIMS OF THE STUDY.......................................................................................................... 29
4 MATERIALS AND METHODS............................................................................................ 30
4.1 Sampling of raw material rainbow trout (I)..................................................................................30
4.2 Rainbow trout roe and its pasteurisation (II, III).........................................................................30
4.2.1 Retail level and fresh roe (II, III) ........................................................................................... 30
4.2.2 Listeria monocytogenes strains (III)....................................................................................... 31
4.2.3 D- and z-value determination (III) ......................................................................................... 31
4.2.4 Pasteurisation of rainbow trout roe (III)................................................................................. 32
4.2.5 Pasteurisation values (III) ...................................................................................................... 32
4.3 Sampling in a fish farm (I ,V) .........................................................................................................33
4.4 Sampling in fish processing factories (IV).....................................................................................33
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4.5 Microbial analyses............................................................................................................................33
4.5.1 Isolation and identification of Listeria spp. and Listeria monocytogenes (I, II, III, IV, V) ... 33
4.5.2 Enumeration of Listeria spp. (II, III) ..................................................................................... 34
4.5.3 Analysis of aerobic, anaerobic, coliform and enterobacteria, fungi and ATP (II, III, IV)..... 34
4.5.4 Identification of bacteria from pasteurised roe (III)............................................................... 35
4.6 Sensory analysis (II, III) ..................................................................................................................35
4.7 Typing of Listeria monocytogenes isolates .....................................................................................35
4.7.1 Ribotyping (I)......................................................................................................................... 35
4.7.2 PFGE-typing (V).................................................................................................................... 35
4.8 Statistical analyses (I, II) .................................................................................................................36
5 RESULTS................................................................................................................................. 37
5.1 Prevalence and location of Listeria monocytogenes in farmed rainbow trout (I) ......................37
5.2 Safety and quality of Finnish retail level roe (II)..........................................................................37
5.2.1 Prevalence of Listeria monocytogenes (II)............................................................................. 38
5.2.2 Microbial and sensory quality (II).......................................................................................... 38
5.3 Pasteurisation of rainbow trout roe (III).......................................................................................39
5.3.1 D- and z-values of four Listeria monocytogenes strain mixture in rainbow trout roe (III).... 39
5.3.2 Microbial quality (III) ............................................................................................................ 39
5.3.3 Sensory quality (III) ............................................................................................................... 40
5.3.4 Prevalence of Listeria monocytogenes (III) ........................................................................... 40
5.3.5 Pasteurisation values (III) ...................................................................................................... 40
5.4 Occurrence of Listeria monocytogenes and Listeria spp. and surface hygiene in fish
processing factories (IV)..................................................................................................................41
5.5 Contamination sources of Listeria monocytogenes at the fish farm (I, V)..................................42
5.6 Distribution of Listeria monocytogenes PFGE-types isolated from different areas of fish
production chain (V)........................................................................................................................44
6 DISCUSSION .......................................................................................................................... 44
6.1 The role of raw fish materials as a source of Listeria monocytogenes (I, V) ..............................44
6.2 Safety and quality of Finnish roe at retail level (II) .....................................................................46
6.3 Safety and quality of pasteurised rainbow trout roe (III) ...........................................................47
6.4 Listeria monocytogenes, Listeria spp. and surface hygiene in fish processing factories (IV)....48
6.5 Sources of Listeria monocytogenes in fish farming (I, V) .............................................................48
7 CONCLUSIONS...................................................................................................................... 51
8 REFERENCES ........................................................................................................................ 53
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ACKNOWLEDGEMENTS
This study was carried out at the Technical Research Centre of Finland, VTT, during the years
1996-2006. The financial support of the Ministry of Agriculture and Forestry, Finnish
Funding Agency for Technology and Innovations (Tekes), Jenny and Antti Wihuri
Foundation and Finnish Food Research Foundation is gratefully acknowledged. The
successful cooperation with the Association of Finnish Fish Retailers and Wholesalers and the
Finnish Fish Farmers’ Association is also warmly acknowledged.
I am very grateful to my supervisors Professor Hannu Korkeala, Dr. Gun Wirtanen and
Dr. Laura Raaska for their valuable advice, guidance and encouraging attitude towards this
thesis.
My special thanks go to my co-authors Anne Arvola, MSc, and Tiina Luoma, MSc, for useful
discussions and help with the sensory analysis and statistical methods. I am grateful for my
co-author Professor Anna-Maija Sjöberg especially for her encouragement at the beginning of
this project. Kaarina Aarnisalo, MScTech, and Satu Salo, MScTech also my co-authors, and
Kirsi Kujanpää, BSc, my roommates during the years of all kinds of research of microbial
safety and hygiene matters are most warmly thanked for the help, problem solving
atmosphere and friendship.
I express my gratitude to Erja Järvinen, Taina Holm, Tarja Vappula, Aila Tuomolin, Raija
Ahonen, Oili Lappalainen and Tarja Niiranen-Jaatinen for the skilful technical assistance and
pleasant cooperation. I wish to thank Dr. Tapani Hattula for introducing the fish research field
to me and Dr. Jaana Mättö and Dr. Maija-Liisa Suihko for the guidance and help in molecular
typing. My gratitude also goes to all the other colleagues at VTT for a pleasant working
atmosphere and assistance.
I am deeply grateful to my parents Erkki and Inger for their ongoing support and help.
Without you, it had been impossible to finish this project. Finally I wish to thank my husband
Juha, for support and love, and our son Vili, for being such an incredible joy of life.
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ABBREVATIONS
AFLP, amplified fragment length polymorphism
ATP, adenosine 5’-triphosphate
aw, water activity
CAMP, Christine-Atkins-Munch-Petersen test, enhanced β-haemolysis test
CFU, colony forming unit
DNA, deoxyribonucleic acid
D-value, decimal reduction time (min)
EB, Listeria enrichment broth
EDTA, ethylenediaminetetraacetic acid
GMP, good manufacturing practices
HACCP, hazard analysis critical control point
ISO, International Organization for Standardization
LMBA, Listeria monocytogenes blood agar
MLST, multilocus sequence typing
MVLST, multi-virulence-locus sequence typing
MEE, multilocus enzyme electrophoresis
NaCl, sodium chloride
PC, plate count
PCR, polymerase chain reaction
PD, potato dextrose
PFGE, pulsed-field gel electrophoresis
p-value, pasteurisation value (min)
RAPD, random amplification of polymorphic DNA
RCM, reinforced clostridial medium
REA, restriction endonuclease analysis
RLU, relative light unit
RTE, ready-to-eat
td, heat death time (min)
TDT, thermal death time
TPB, tryptic phosphate broth
TBE, tris-borate-ethylenediaminetetraacetic acid
TE, tris-ethylenediaminetetraacetic acid
Tref, reference temperature
UPGMA, unweighted pair group method using arithmetic averages
VTT, Technical Research Centre of Finland
z-value, degrees required for the thermal destruction curve to traverse one log cycle
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ABSTRACT
Contamination of fish products with Listeria monocytogenes, a bacterium causing listeriosis,
presents a risk to consumer health. To better control and prevent this contamination, the
present study investigated the prevalence and sources of L. monocytogenes in different stages
of fish production chain as well as the effects of a pasteurisation method on safety of rainbow
trout roe products.
Farmed rainbow trout from different fish farms were found to contain L. monocytogenes and
Listeria spp. at an average rate of 9% and 14%, respectively. L. monocytogenes prevalence
varied greatly among different fish farms from 0 to 75%. The location of L. monocytogenes
and Listeria spp. in different parts of the rainbow trout differed significantly (p < 0.0001).
L. monocytogenes contamination in rainbow trout occurred almost exclusively in the gills
(96%) and only sporadically in the skin and viscera. Special effort, during the transportation
and processing of raw fish, should be focused on the isolation and removal of rainbow trout
gills before L. monocytogenes contamination spreads further.
Presence of L. monocytogenes in different Finnish fish species roe products in retail markets
varied between 2 to 8%. Recovery of L. monocytogenes was significantly (p < 0.01) higher in
fresh-bought roe products (18%) than in frozen (0%) and frozen-thawed (2%) roe products. In
terms of aerobic and coliform bacteria the microbial quality of roe samples was poor in 57%
and 73% of the samples, respectively, and 20% of the samples were unacceptable to taste.
Pasteurisation, at 62 °C or at 65 °C for 10 minutes, of rainbow trout roe eliminated all
inoculated 8 log units of L. monocytogenes. Based on the determined D- and z-values, for four
L. monocytogenes strain mixtures, these pasteurisations theoretically destroyed 46 and 154 log
units of L. monocytogenes cells, respectively. The quality of pasteurised vacuum packaged
rainbow trout roe was found to be consistently good, in terms of microbial as well as sensory
quality, for up to six months stored at 3 °C.
L. monocytogenes and Listeria spp. appeared on cleaned surfaces of one-third and two-thirds
of the 23 studied fish factories, at least sporadically. The presence of Listeria spp. on the
factory surfaces was indicative of increased possibility of occurrence in the fish products. In
factories where Listeria spp. was found on surfaces they were often (10/13) found in some
products. The overall L. monocytogenes contamination level of different ready-to-eat fish
products from the fish factories varied from 0 to 20%.
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The main L. monocytogenes contamination sources in the studied fish farm were the brook and
river waters, as well as other runoff waters from environment. Rainy weather conditions were
found to increase the probability of finding L. monocytogenes in the fish farm environment
and in fish. The L. monocytogenes contamination in fish gradually disappeared over several
months. Such disappearance, however, was faster in the surrounding sea water than in the fish.
Presence of certain L. monocytogenes pulsed-field gel electrophoresis (PFGE) -types, after the
first discovery months earlier in some other sample type, was typical for sea bottom soil
samples. The fish farm studied did not spread L. monocytogenes contamination, but suffered
from L. monocytogenes contamination from environmental sources.
The PFGE-typing of L. monocytogenes isolates, from 15 fish factories, showed that the same
pulsotypes of L. monocytogenes occurred in isolates of final fish products as well as both raw
fish and fish production environment isolates. Thus, raw fish materials and production
environment and machines are sources of L. monocytogenes contamination that both need to
be properly controlled.
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LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following papers referred to in the text by Roman numerals I to V:
I
Miettinen, H. and Wirtanen, G. 2005. Prevalence and location of Listeria
monocytogenes in farmed rainbow trout. Int. J. Food Microbiol. 104, 135-143.
II
Miettinen, H., Arvola, A., Luoma, T. and Wirtanen, G. 2003. Prevalence of Listeria
monocytogenes in, and microbiological and sensory quality of, rainbow trout, whitefish
and vendace roes from Finnish retail markets. J. Food Prot. 66, 1832-1839.
III
Miettinen, H., Arvola, A. and Wirtanen, G. 2005. Pasteurization of rainbow trout roe:
Listeria monocytogenes and sensory analyses. J. Food Prot. 68, 1641-1647.
IV
Miettinen, H., Aarnisalo, K., Salo, S. and Sjöberg, A.-M. 2001. Evaluation of surface
contamination and the presence of Listeria monocytogenes in fish processing factories.
J. Food Prot. 64, 635-639.
V
Miettinen, H. and Wirtanen, G. 2006. Ecology of Listeria spp. in a fish farm and
molecular typing of L. monocytogenes from fish farming and processing companies. Int.
J. Food Microbiol. In press.
The original papers have been reprinted with kind permission from Elsevier (I, V) and Journal
of Food Protection (II, III, IV).
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1 INTRODUCTION
The Gram-positive bacterium Listeria monocytogenes was probably first recognised in two
rabbits in Sweden 1910 (Hülphers 1911). Over a decade later Murray et al. (1926) in the United
Kingdom and Pirie (1927) in South Africa recognised a disease in laboratory rabbits, guineapigs and gerbils caused by a Gram-positive bacillus. Pirie named the causative bacterium
Listerella hepatolytica and Murray, Webb and Swann named the bacterium Bacterium
monocytogenes as it produced large mononuclear leucocytosis. The genus name Listeria was
given by Pirie (1940) as the genus name Listerella was already used.
Recognition of L. monocytogenes as a significant food borne pathogen occurred only in the
early 1980’s, with demonstration of food borne listeriosis outbreak (Schlecht et al. 1983).
L. monocytogenes is widely distributed in the environment and occurs in almost all food raw
materials from time to time. The disease listeriosis usually occurs in high-risk groups, including
pregnant women, neonates and immunocompromised adults, but may occasionally occur in
persons who have no predisposing underlying condition. Listeriosis is one of the most severe
food borne infections, with low morbidity but high mortality 30% (Rocourt et al. 2001). The
yearly medical costs and productivity losses from the acute illness from food borne Listeria in
the USA are estimated to be one, two and three times the costs caused by Salmonella spp.,
Campylobacter spp. and Escherichia coli O157:H7, respectively, despite the prevalence of
these diseases being over 500, 700 and 25 times the number of listeriosis cases, respectively
(Anonymous 2000a).
L. monocytogenes is able to multiply in high salt concentrations even at refrigerated temperatures
with or without oxygen. It is resistant to diverse environmental conditions and it can survive in
industrial environments for years regardless of cleaning procedures (Rocourt et al. 2001,
Hoffman et al. 2003). L. monocytogenes is ubiquitous in nature and therefore also aquatic
creatures are potential bacterium sources. A part of the seafood products undergo various
processing steps that inactivate the bacterium, if present on the raw product. L. monocytogenes
cross-contamination of products after listericidal processing, however, presents a major problem
especially for ready-to-eat products. Furthermore, there are seafood products that are eaten raw,
without any listericidal step, like cold-smoked and cold-salted fish. A lot of work has been
conducted to study the sources of L. monocytogenes as well as means to control its growth and
contamination in different food sectors (Chasseignaux et al. 2002, Pak et al. 2002,
Gudbjörnsdóttir et al. 2004, Thimothe et al. 2004). More research is still needed on focused risk
areas, including seafood processing, to prevent problems caused by L. monocytogenes.
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2 REVIEW OF THE LITERATURE
2.1 Listeria monocytogenes
2.1.1 Listeria monocytogenes
L. monocytogenes is a small 0.5 µm in diameter and 1 to 2 µm in length, regular Grampositive rod with rounded ends. Cells are found either singly, in short chains, arranged in V
and Y forms or in palisades. Sometimes cells are coccoid and average about 0.5 µm in
diameter, causing them to be confused with streptococci (Rocourt 1999). L. monocytogenes is
facultatively anaerobic. It is motile because of its few peritrichous flagella when cultured at
20 °C to 25 °C. The bacterium does not form spores or capsules (Seeliger and Jones 1986). Its
optimum growth temperature is between 30 °C and 37 °C and temperature limits for growth
are -0.4 °C to 1 °C and 45 °C to 50 °C (Seeliger and Jones 1986, Golden et al. 1988, Junttila
et al. 1988, Walker et al. 1990). The G + C content of the DNA is 36% to 38%.
L. monocytogenes is widely distributed in nature and is found in water, mud, sewage,
vegetation and in the faeces of animals and humans (Seeliger and Jones 1986). The genus
Listeria includes six species: L. gray, L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri
and L. welshimeri (Rocourt 1999).
2.1.2 Listeriosis
Listeriosis is a disease caused by bacteria of the genus Listeria. L. monocytogenes is the
pathogenic species in both animals and humans (McLauchlin and Jones 1999). Few cases of
human infections, however, are caused by L. ivanovii (Cummins et al. 1994, Lessing et al.
1994) and L. seeligeri (Rocourt et al. 1986).
Principally listeriosis causes intra-uterine infection, meningitis and septicaemia. Listeriosis
during pregnancy manifests as a severe systemic infection in the unborn or newly delivered
infant as well as a mild influenza-like bacteraemic illness in the pregnant woman. Pregnancy
and neonatal cases comprise 10% to 20% of the listeriosis cases (McLauchlin et al. 2004). In
adults and juveniles, the main presentations are as central nervous system infection and/or
septicaemia. Most adult and juvenile cases occur amongst the immunosuppressed (Table 1),
e.g. patients receiving steroid or cytotoxic therapy or with malignant neoplasms. Other groups
include patients with AIDS, diabetics, individuals with prosthetic heart valves or replacement
joints and individuals with alcoholism or alcoholic liver disease. The incidence of infection
increases with age, with the mean age of adult infection being over 55 years (McLauchlin et
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al. 2004). Mild non-invasive listeriosis, with gastroenteritis and fever, has also been reported
in otherwise healthy individuals (Riedo et al. 1994, Miettinen et al. 1999, Aureli et al. 2000).
Table 1. People at risk for listeriosis (Hof 2003).
Population
Incidence of listeriosis in some at risk populations
(per 100000 individuals per year)
Normal population
Aged persons (>70 years)
Alcoholics
Diabetic people
Iron overload
Pregnant women
Cancer patients
Steroid therapy
Lupus erythematodes
Kidney transplant recipients
Chronic lymphatic leukaemia
AIDS
Leukaemia (acute monocytic and acute lymphoblastic)
0.7
2
5
5
5
12
15
20
50
100
200
600
1000
Although Murray et al. (1926) first suspected an oral route for the bacterial infection observed
in animals in 1924, it was not until 1981 that an outbreak of listeriosis in Canada was linked
to a contaminated food source (Schlecht et al. 1983). Listeriosis can occur sporadically or
epidemically; in both, contaminated foods are the primary vehicles of transmission. Unlike
infection by other common food borne pathogens, such as Salmonella which rarely results in
fatalities, listeriosis is associated with a mortality rate of approximately 20% to 40% (Farber
and Peterkin 1991). Listeriosis is, however, a rare disease (Gerner-Smidt et al. 2005), despite
of the relatively frequent exposure to the causative bacterium. An average of five to nine
exposures to L. monocytogenes occur per person per year (Grif et al. 2003). The
asymptomatic point prevalence of faecal carriage of L. monocytogenes is from 0 to 21% and a
cumulative prevalence as high as 77% in high-risk groups, e.g. household contacts of people
with listeriosis (Slutsker and Schuchat 1999). The incubation period and infective dose have
not been firmly established. Reported incubation times vary from one day to three months
(Linnan et al. 1988). The infective dose has so far been reported to be relatively high (>103
CFU/g) (McLauchlin 1996) or the infection has been suggested to be caused by a prolonged
daily consumption of food contaminated with L. monocytogenes (101–105 CFU/g) (Maijala et
al. 2001). The attack rate of various strains, including outbreak and sporadic listeriosis strains,
is also likely to be low (McLauchlin et al. 2004) explaining the rare incidence <1/100000
inhabitants (Anonymous 2004). In Finland, the yearly incidence rates have varied between
0.35 and 1.0/100000 inhabitants since 1995 (Anonymous 2006). A number of food items
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including coleslaw (Schlech et al. 1983), pasteurized milk (Fleming et al. 1985), different
cheeses (Linnan et al. 1988, Goulet et al. 1995, Anonymous. 2001, Makino et al. 2005), pâté
(McLauchlin et al. 1991), rillettes (Goulet et al. 1998, de Valk et al. 2001), rice salad
(Salamina et al. 1996), chocolate milk (Dalton et al. 1997), corn and tuna salad (Aureli et al.
2000), hot dogs and delicatessen meats (Anonymous 1998b, Lin et al. 2006), butter
(Lyytikäinen et al. 2000) and turkey (Anonymous 2000b, Frye et al. 2002) have caused
listeriosis outbreaks. Some outbreaks have also been connected to the consumption of seafood
(Table 2). In addition cold-smoked fish in Finland (Lukinmaa et al. 2003) and seafood in Norway
and Sweden (Loncarevic et al. 1998, Rørvik et al. 2000) have been suggested causing listeriosis.
Table 2. Listeriosis outbreaks connected with seafood consumption.
Suspected seafood
No. of cases
(deaths)
Symptoms
Country,
area
References
Shellfish and raw fish
22(6)
Premature labour, fetal distress,
respiratory symptoms,
meningitis, flu-like illness,
urinary tract symptoms,
diarrhoea, vomiting
New
Zealand
Lennon et al. 1984
Fish
1(0)
Meningitis
Italy
Facinelli et al. 1989
Smoked mussels
3(0)
Vomiting, diarrhoea
Tasmania
Mitchell 1991,
Misrachi et al. 1991
Shrimps
11(1)
Fever, nausea, vomiting,
musculoskeletal symptoms,
diarrhoea, fetal demise
USA
Riedo et al. 1994
Cold-salted ‘gravad’
rainbow trout
8(2)
Amniotitis, meningitis,
premature birth, fever,
septic arthritis, septicaemia
Sweden
Ericsson et al. 1997
Smoked mussels
3(0)
Perinatal lethargy,
malaise
New
Zealand
Brett et al. 1998
Cold-smoked rainbow trout 5(0)
Fever, vomiting, fatigue,
arthralgia, headache
Finland
Miettinen et al. 1999
Imitation crabmeat
Diarrhoea, cramps,
fever, projectile vomiting,
nausea
Canada
Farber et al. 2000
2(0)
2.1.3 Isolation and identification
The first isolation methods were generally based on the direct culture of samples on simple
agar media. Isolation was difficult and inoculation into test animals was recommended in case
of low numbers of viable Listeria cells. At the end of 1930 the first discoveries of the
7
refrigeration temperatures resulted in more positive samples compared to incubation at
elevated temperatures (Beumer and Hazeleger 2003). Ten years later Gray et al. (1948)
described a new technique based on cold enrichment at 4 °C for several weeks. The main
disadvantage of the method was the long incubation period, up to several months. Modern
isolation methods are based on one or two-step enrichment with a variety of selective agents,
followed by plating on selective plating media (Anonymous 1996, 1999, 2005, Hitchins
2003). Quantitative and semiquantitative methods are used (Anonymous 1998a, 1999) in
addition to detection of L. monocytogenes. The selective agents for background flora
inhibition include acriflavine, cyclohezimide, cefotetan, ceftazidime, colistin, fosfomycin,
lithium chloride, nalidixic acid, potassium thiocyanate, and polymyxin B-sulphate.
Enrichments are performed at 30-37 °C for one or two days. Solid media used for isolation of
Listeria spp. contain selective agents and indicator substrates e.g. blood or chromogens to
distinguish Listeria spp. from background flora or from different Listeria spp. The
confirmation of presumptive L. monocytogenes colonies on the selective media is performed
with Gram-staining, catalase reaction, motility at 25 °C, β-haemolysis test, fermentation of
rhamnose and xylose and CAMP-test. Several commercial tests developed for identification
of L. monocytogenes exist as alternatives to conventional testing. The results of various
isolation processes of L. monocytogenes differ somewhat with different isolation methods
depending on selective compounds, presence of Listeria spp. and background flora in the
sample, and possible presence of injured Listeria cells in the sample (Johansson 1998, Duarte
et al. 1999, Vaz-Velho et al. 2001, Pinto et al. 2001, Cornu et al. 2002, Gnanou Besse 2002).
Methods can also bias the results and proportions of different Listeria spp. (Bruhn et al. 2005,
Gnanou Besse et al. 2005).
With or without isolation and further possibility of subtyping, rapid detection of Listeria spp.
and L. monocytogenes is sometimes needed. Clinical and even food specimens can be
analysed for the presence of Listeria spp. or L. monocytogenes from selective enrichment
broths with immunoassays using commercial antibodies. Also available are DNA
hybridization probes and PCR assays for food and environmental specimens, both for
research and commercial use (Allerberger 2003, Gasanov et al. 2005). Using PCR
L. monocytogenes has been detected e.g. in smoked salmon (Simon et al. 1996, Becker et al.
2005), in cold salted (gravad) rainbow trout (Ericsson and Stålhandske 1997), in salmon and
salmon products (Norton et al. 2001, Rodríguez-Lázaro et al. 2005), channel catfish (Wang
and Hong 1999), fish seafood products (Bansal et al. 1996, Gouws and Liedemann 2005) and
environmental samples (Norton et al. 2001).
8
2.1.4 Subtyping
Subtyping of closely related L. monocytogenes strains is needed to be able to confirm
outbreak sources, establish transmission patterns and determine and monitor epidemic strain
reservoirs. A wide range of various pheno- and genotyping methods are available for typing
of microbes. Genotyping is based on the assumption that strains of the causal organism
isolated from different sources are clonally related, and are similar or identical in their
phenotypic and molecular characteristics. Therefore, these methods are based on
specie-specific proteins or genes that are relatively stable over time and are passed on from
generation to generation (Gasanov et al. 2005). A summary of typing methods often used and
those that are likely to be used more in the future for subtyping of L. monocytogenes are
presented in Table 3.
2.2 Growth characteristics of Listeria monocytogenes
The viable populations of L. monocytogenes start to decrease at the temperatures above 50 °C
(Golden et al. 1988). Walker et al. (1990) studied the minimum growth temperature and found
that there was slow growth at a temperature range of -0.1 °C to -0.4 °C for three
L. monocytogenes strains. Also a notable variation in the growth among different strains of
L. monocytogenes is apparent, especially at refrigeration temperatures (Barbosa et al. 1994,
Begot et al. 1997). The generation time of 39 L. monocytogenes strains at 4 °C and 10 °C
varied between 24.6 to 69 h and 3.5 to 8.6 h, respectively (Barbosa et al, 1994).
L. monocytogenes survives freezing well and the frozen storage causes a limited reduction in
the viable population of L. monocytogenes (Lou and Yousef 1999). The low pH of the frozen
media, like tomato soup (pH 4.7), compared to other foods (ground beef, turkey, frankfurters,
canned corn and ice-cream mix) increased the death and injury of L. monocytogenes cells
during frozen storage (Palumbo and Williams 1991). Harrison et al. (1991) found a less than
three log unit decrease in inoculated L. monocytogenes counts in fish and shrimps after
freezing at -20 °C for three months. Slow freezing at -18 °C is more lethal and injurious than
rapid freezing at -198 °C (El-Kest et al. 1991).
L. monocytogenes grows at pH 4.3 to 9.2 (Farber et al. 1989, Parish and Higgins 1989, Petran
and Zottola 1989). It has, however, the ability to adapt and survive at even lower pH (3 to 3.5)
(O´Driscoll et al. 1996, Shabala et al. 2002, Liu et al. 2005) and higher pH (12) (Liu et al.
2005). Stationary phase cells survive better than exponential phase cells and glucose added to
the media helps to protect the bacteria by providing energy and metabolic precursors (Shabala
9
Table 3. Subtyping methods for L. monocytogenes.
10
Method
Principle
Comments
References
Serological typing
Based on antibodies that specifically react with somatic (O)
antigens and flagellar (H) antigens of Listeria species.
First-level subtyping. Thirteen serotypes:
1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e
and 7. Most of the clinical and food related isolates
belong to three serotypes: 1/2a, 1/2b and 4b.
Seeliger and Höhne 1979
McLauchlin 1990
Schönberg et al. 1996
Phage typing
Based on the specific interaction of a particular bacteriophage
with its host strain, resulting in host cell lysis.
Able to process relatively large numbers of
cultures with good discrimination power.
Not all strains are typable, not always reproducible.
Rocourt et al. 1985
McLauchlin et al. 1996
Multilocus enzyme
electrophoresis
(MEE)
Differentiates isolates according to the electrophoretic
mobility of a large number of strains metabolic enzymes.
Electromorph profiles (electrophoretic types, ETs) index
the whole chromosomal genome.
Discriminatory power of the method is relatively low.
ETs distinguished by MEE are more stable than typed
strains of many genotyping methods.
Used in several seafood studies.
Rørvik et al. 1995, 2000
Caugant et al. 1996
Flint and Kells 1996
Boerlin et al. 1997
Ribotyping
Strains are characterized for restriction fragment length
polymorphisms associated with ribosomal operon(s).
Chromosomal DNA is digested with restriction enzyme
followed with hybridisation using labelled
16S+23S rRNA or rDNA probe.
Less discriminating than bacteriophage typing,
MEE or REA. The reproducibility and typeability
are good. Suited for long-term epidemiological or
phylogenetic studies. Widely used for tracking
and subtyping in seafood factories with mostly
EcoRI as the restriction endonuclease.
Grimont and Grimont 1986
Stull et al. 1988
Nørrung and Gerner-Smidt 1993
Graves et al. 1999
Norton et al. 2001
Thimothe et al. 2004
Restriction enzyme
analysis
(REA)
Restriction enzymes recognize and cut particular sequences
within DNA molecules producing a banding pattern of
fragments with varying sizes separated and visualised
with gel electrophoresis.
Universally applicable, sensitive, cost effective and
easy to do analysis. Limitation is in the difficulty of
comparing the complex profiles which consist of
hundreds of bands. Exploited e.g. in seafood
outbreak study and in epidemiological survey in
seafood-processing plants.
Ericsson et al. 1997
Graves et al. 1999
Rørvik et al. 2000
Gasanov et al. 2005
Pulsed-field gel
electrophoresis
(PFGE)
The intact bacteria are digested using one or more
restriction endonucleases that cut infrequently. Large
chromosomal DNA fragments are separated by pulsedfield gel electrophoresis.
Discriminating and reproducible. Time (2 to 3 days)
needed to complete the analysis is long. Used in
several L. monocytogenes surveys in seafood industry
and in listeriosis outbreak studies caused by seafood.
Destro et al. 1996
Brett et al. 1998
Graves et al. 1999
Olive and Bean 1999
Johansson et al. 1999
Table 3. continued
11
Method
Principle
Comments
References
Random amplified
polymorphic DNA
(RAPD)
Genomic DNA is characterized based on the number and
size of amplified DNA fragments generated by a single
random or universal primer in a PCR.
Small changes in the genomic DNA will result in different
sizes and numbers of amplified fragments.
Rapid and relatively simple technique.
High discriminatory power, screen large number of
samples. Suitable for epidemiological typing. Patterns
have inconsistent reproducibility. Well-standardised
RAPD protocol needed to obtain reliable results.
Used in surveys in seafood industry and in listeriosis
outbreak studies caused by seafood.
Destro et al. 1996
Wernars et al. 1996
Graves et al. 1999
Farber et al. 2000
Fonnesbech Vogel et al. 2001
Mędrala et al. 2003
Gasanov et al. 2005
Amplified fragment DNA is digested using restriction enzyme(s), followed by
length polymorphism the ligation of the resulting fragments to oligonucleotide
(AFLP)
adapter complementary to the base sequence of the restriction
site. The adapters are designed so that the original restriction
site is not restored after ligation, thus preventing further
restriction digestion. Selective amplification by PCR of sets of
these fragments is achieved using primers corresponding to the
contiguous base sequences in the adapter, restriction site plus
one or more nucleotides in the original target DNA.
High discriminatory power, excellent typeability
and high reproducibility .
Can be successfully applied to analyse
L. monocytogenes routes and ecology in food
processing industry.
Vos et al. 1995
Guerra et al. 2002
Autio et al. 2003
Fonnesbech Vogel et al. 2004
Multilocus sequence Uses automated DNA sequencing to characterize the alleles
typing
present at different housekeeping genes.
(MLST)
Also targeted to hypervariable genes.
Highly discriminatory and provides unambiguous
result. Differentiate most of the strains better than
or equally to PFGE.
Hypervariable genes showed low degree of
discrimination.
Enright and Spratt 1998
Salcedo et al. 2003
Multi-virulencelocus sequence
typing (MVLST)
Targets virulence and virulence associated genes.
Revazishvili et al. 2004
Meinersmann et al. 2004
Virulence associated genes showed higher
Zhang et al. 2004
discriminatory power than ribotyping, PFGE and
Chen et al. 2005
MLST. MLST and MVLST are valuable typing
methods for the future after identification of the
applicable discriminatory genes and the number of gene
loci that provide optimal resolution. Results can be
compared via e.g. worldwide web that assists the
observation of global listeriosis epidemics and sources.
et al. 2002). The induction of an acid tolerance response can provide cross-protection against
thermal stress, ethanol, osmotic stress, or stress due to a surface-active agent. It has also been shown
that acid tolerant strains display increased virulence relative to that of the wild type (O’Driscoll et
al. 1996).
L. monocytogenes grows optimally at aw above 0.97 (Petran and Zottola 1989), however, it has a
rather unique ability to multiply at aw values as low as 0.90 (Lou and Yousef 1999). It can survive
for extended periods at even lower aw values (Shahamat et al. 1980). The bacterium also endures
high salt concentrations 25.5 % NaCl (Shahamat et al. 1980) and has been isolated e.g. from brine
used in fish industry (Jemmi and Keusch 1994, Autio et al. 1999, Norton et al. 2001,
Gudmundsdóttir et al. 2005).
In addition to the above mentioned general growth parameters plenty of additional factors exist
effecting the growth, survival and adaptation of different L. monocytogenes strains like inoculum
size, growth medium with inhibitory as well as protective compounds and structure, atmosphere,
and pre-incubation conditions. All these factors also have a combined effect that can not always be
predicted based on results tested with an individual variable.
2.3 Thermal resistance of Listeria monocytogenes
The heat resistance of L. monocytogenes is influenced by many factors such as strain variation,
previous growth conditions, exposure to heat shock, acid, as well as other stresses, and composition
of the heating menstruum (Doyle et al. 2001). D-value is used to describe the heat resistance of a
certain strain at a certain temperature as it is the time needed to destroy 90% of cells at that
temperature. z-value is the temperature difference required to destroy 90% of the bacteria with a 10fold change in heating time. Table 4 presents D- and z-values for some L. monocytogenes strains in
different seafood. The strain variation of 21 L. monocytogenes strains at 55°C in BHI broth at pH 6
was 4.7 fold (23.8 to 111 min) shown by the D55°C-values (de Jesús and Whiting 2003). Golden et
al. (1988) showed that a L. monocytogenes isolate from brie cheese had 1.5 to 2.8 fold higher D56value than the three listeriosis outbreak strains tested.
Cells in stationary phase of growth appear to be the most resistant to thermal stress (Pagán et al.
1998, Doyle et al. 2001). Stationary phase cells of L. monocytogenes had 2.8 to 5.6 times higher
D60°C-values (0.45 to 12.5 min) than the logarithmic phase cells, each tested in three different media
12
Table 4. L. monocytogenes D- and z-values for seafood products and sea water.
Seafood
Strain
(No.)
Salmon
Salmon
Cod
Cod
Imitation crabmeat1
Imitation crabmeat2
Crabmeat
Lobster
Crawfish
Mussels
Salmon caviar5
Salmon caviar6
Sea water7
Sea water7,8
D-values (min) at temperature (°C) of
55
062 (1)
057 (1)
062 (1)
057 (1)
Mixture (4)
Mixture (4)
ScottA (1 )
12.0
Mixture (5)
Mixture (3)
10.23
Mixture (7)
L. innocua (1)
L. innocua (1)
ScottA, KM (2)
ScottA, KM (2)
58
60
62
10.73
8.48
7.28
6.18
9.7
10.2
4.48
4.23
1.98
1.95
2.07
3.02
0.87
8.33
16.25
1.36
2.78
63
65
0.87
1.18
0.28
0.27
2.1
2.3
2.61
2.39
1.98
5.49
2.97
3.55
0.69
1.15
66
0.4
0.4
1.064
0.19
1.85
0.77
0.85
0.58
0.64
13
1
0.40
0.41
z-value References
68
70
(°C)
0.2
0.22
0.15
0.13
0.07
0.17
0.03
0.05
5.6
6.7
5.7
6.1
5.8
5.7
8.4
5.0
5.5
4.25
5.7
5.3
10.8
6.27
Ben Embarek and Huss 1993
Ben Embarek and Huss 1993
Ben Embarek and Huss 1993
Ben Embarek and Huss 1993
Mazzotta 2001
Mazzotta 2001
Harrison and Huang 1990
Budu-Amoako et al. 1992
Dorsa et al. 1993
Bremer and Osborne 1995
Al-Holy et al. 2004
Al-Holy et al. 2004
Bremer et al. 1998
Bremer et al. 1998
Stationary phase cells, 2Salt adapted cells (5 h, 15% NaCl), 357.2 °C, 462.7 °C, 5Aluminum TDT tubes, 6Glass TDT tubes, 7Filter sterilized (0.22 µm), 8Sea water adapted
cells (7d)
13
(minced beef, tryptic phosphate broth TPB and TPB supplemented with 8 g/l lactic acid) at
four different pH from 5.4 to 7.0 (Jørgensen et al. 1999). L. monocytogenes Scott A strain had
7.6 times higher D56°C-value at the early stationary phase than at the exponential phase (Lou
and Yousef 1996).
The composition of the growth medium, whether a food or laboratory culture broth, affect
rates of growth and the synthesis of cellular constituents that determine the thermal tolerance
of bacterial cells (Doyle et al. 2001). The D60°C-values for L. monocytogenes 13-249 were 2 to
6 fold higher in minced beef than in TPB (Jørgensen et al. 1999). In half cream, double cream
and butter the D-values of two L. monocytogenes strains were 1.1 to 8 fold higher than in TSB
also indicative of notable differences in D-values between food products (Casadei et al. 1998).
The environment in which cells are grown can be a major determinant of their heat resistance.
L. monocytogenes –strain Scott A growing in tryptic phosphate broth containing 0.09, 0.5, 1.0
or 1.5 M NaCl was heated in media with the same salt concentrations resulting in 4 log
reduction at 60 °C in 1.6, 2.5, 7.4 and 38.1 min, respectively (Jørgensen et al. 1995).
Increased heat resistance was also induced by starvation, low pH, and addition of
antimicrobial compounds like ethanol, or hydrogen peroxide to the growth media (Lou and
Yousef 1996). The growth temperature affects the heat resistance and in general the cells
grown at higher temperatures are more heat resistant than those grown at lower temperatures
(Smith et al. 1991, Pagán et al. 1998). The rate at which cells are heated during testing also
influences their survival. When cells are heated slowly, they exhibit a greater heat resistance
than when heated rapidly (Kim et al. 1994, Hassani et al. 2005).
Heat-shock, a short-term exposure of cells to temperatures above the optimum growth, results
in increased heat resistance. The degree of enhanced thermal resistance is strain dependent
and also varies with the length of the heat shock, the pH of the medium, and the growth phase
of the cells. The maximum increase in thermotolerance was 4 and 7 fold for a
L. monocytogenes strain previously grown at 37 °C and 4 °C and heat-shocked at 45 °C and at
47.5 °C, respectively (Pagán et al. 1997). On the other hand cold-shock decreases the heat
resistance of L. monocytogenes (Miller et al. 2000). Increased heat tolerance can also be
induced by short-term exposure to high salt (Jørgensen et al. 1995) or solute levels (Smith and
Hunter 1988). Decreasing aw values and increasing solute concentrations result in greater heat
resistance in L. monocytogenes (Doyle et al. 2001). A systemic approach to determine global
thermal inactivation parameters for various food pathogens resulted with only a limited
number of factors having a significant effect (p < 0.05) on the D-value. The collected D-value
14
data (n=967) for L. monocytogenes showed that the presence of 10% salt or aw<0.92 resulted
in a high heat resistance and it became the most heat resistant vegetative pathogen (van Asselt
and Zwietering 2005). This did not mean that other effects do not occur, but that the low aw
was statistically significant based on the data studied.
2.4 Listeria monocytogenes in nature, seafood industry and seafood products
L. monocytogenes is ubiquitous in nature and therefore aquatic creatures are also potential
sources of the bacterium. Part of the seafood products undergoes various processing steps that
can inactivate the bacterium if present on the raw product. L. monocytogenes, however, can
also enter the product both during and after processing due to poor sanitation conditions or
inadequate manufacturing practices (Jinneman et al. 1999).
2.4.1 Occurrence of Listeria monocytogenes in environment
Forest soil, cultivated and uncultivated fields, mud, feed, feeding grounds, wildlife faeces and
birds (Weis and Seeliger 1975) have been found to be substantially (8.4 to 44%) contaminated
with Listeria spp. Cultivated land is more often contaminated with L. monocytogenes than
uncultivated land (Weis and Seeliger 1975, Dowe et al. 1997). The soil type influences the
survival of L. monocytogenes, sandy soil yields a lower level and clay loam and sandy loam
soils a higher count (Dowe et al. 1997). Soil has been suggested to be the natural reservoir of
Listeria spp. since it is able to multiply there (Botzler et al. 1974, Dowe et al. 1997).
L. monocytogenes has also been found in sludge from a fish farm (Jemmi and Keusch 1994).
Water environments such as coastal sea waters and rivers containing a high organic load have
been found to carry Listeria spp. In California, river and costal sea waters contained Listeria
spp. in 81% and L. monocytogenes in 62% of the samples (Colburn et al. 1990). In Scotland,
L. monocytogenes appeared throughout the course of a river passing through areas ranging
from sparsely populated mountains to highly populated urban areas (Fenlon et al. 1996). In a
northern province of the Netherlands, L. monocytogenes contamination occurred in 37% of
the surface waters of canals and lakes and 67% of water containing effluent from a sewage
treatment plant (Dijkstra 1982). In Italy, L. monocytogenes was found in 40%, 58% and 67%
of river, treated and untreated sewage water, respectively (Bernagozzi et al. 1994). Most of
the treated water (84.4%) and raw sludge (89.2%) was contaminated with Listeria spp. in six
French urban wastewater treatment plants (Paillard et al. 2005). In Sweden, 12% of raw
sludge samples were contaminated with L. monocytogenes, however, contamination occurred
in only 2% of the treated sludge samples (Sahlström et al. 2004). The presence of
15
L. monocytogenes in ground or spring water is rare, however, some cases have been reported
(Korhonen et al. 1996, Schaffer and Parriaux 2002). Although L. monocytogenes has been
found in sea water several times, there are sea areas where it has not been detected (Dijkstra
1982, Rørvik et al. 1995, Ben Embarek et al. 1997). The survival of L. monocytogenes in
water depends on many factors, but it has been found to survive several weeks in suitable
water environments (Botzler et al. 1974, Bremer et al. 1998). Hsu et al. (2005) showed that
the two L. monocytogenes strains studied did not readily survive and compete with the marine
flora in sea water or in salmon blood-water in elevated (>7 °C) temperatures.
A large proportion of faecal samples collected from healthy animals with no clinical symptoms of
listeriosis may contain L. monocytogenes (Wesley 1999). It is also of no surprise that different
kinds of fish, squid and crustaceans have been found to contain L. monocytogenes (Table 5). The
L. monocytogenes contamination in salmon, the most studied fish species, also varied widely in the
literature (0 to 88%, Table 5). Contamination of other species and shellfish with L. monocytogenes
was also significant from 0 to 51%. Differences, however, occur in sample collection and
transportation, sampling methods, and actual analyses all which may influence the reported
results. The mean L. monocytogenes prevalence of all studies was 14% (Table 5).
2.4.2 Sources and routes of Listeria monocytogenes contamination and its occurrence in
seafood industry
Contamination of final products by L. monocytogenes in seafood processing plants may occur
from various sources. In addition to frequent contamination of raw materials (Table 5), other
sources are also significant in terms of final product safety. Table 6 summarises
L. monocytogenes contamination of environment, raw materials, products during processing
and final products in different fish processing factories.
Effective cleaning was found to be an essential preventive measure in reducing the amount of
L. monocytogenes contamination in fish processing (Jemmi and Keusch 1994, Rørvik et al.
1997, Fonnesbech Vogel et al. 2001, Hoffman et al. 2003). Many times the procedures used for
cleaning and disinfection were, however, insufficient in removing persistent L. monocytogenes
contamination in fish processing factories (Fonnesbech Vogel et al. 2001, Norton et al. 2001,
Dauphin et al. 2001, Gudbjörnsdóttir et al. 2004, Thimothe et al. 2004, Gudmundsdóttir et al.
2005). In most of the studied fish processing factories, one or a few L. monocytogenes clones
were found to persist for several months or years in the processing environment despite the
normal washing regime (Johansson et al. 1999, Fonnesbech Vogel et al. 2001, Dauphin et al.
16
Table 5. Prevalence of Listeria spp. and L. monocytogenes in live seafood, fresh seafood in retail markets and in fresh raw seafood material from processing
factories.
17
Seafood type (country, area)
Sampling location
Specification
Salmon (Norway)
Salmon (Norway)
Salmon1 (Norway)
Salmon2 (Norway, Faroe Islands)
Salmon (Norway)
Salmon1 (Norway)
Salmon (Norway)
Salmon (UK)
Salmon (UK)
Salmon1 (USA)
Salmon1 (USA)
Salmon1 (USA)
Salmon1 (USA)
Salmon1 (USA)
Salmon1 (USA)
Salmon1 (USA)
Salmon1 (USA)
Salmon (Chile)
Salmon trout (Portugal)
Trout (Portugal)
Seatrout (Norway)
Trout (UK)
Rainbow trout (Finland)
Rainbow trout (Finland)
Rainbow trout1 (Finland)
Rainbow trout (Spain)
Rainbow trout (Switzerland)
Rainbow trout (Switzerland)
Rainbow trout (Switzerland)
Rainbow trout (USA)
Brown trout (Spain)
Pike (Spain)
Whiting (France)
Live, farmed
Processing factory
Processing factories
Two processing factories
Processing factory
Producer
Processing factory
Commersial outlets
Processing factory
Two processing factories
Freezer warehouse
Freezer warehouse
Freezer warehouse
Freezer warehouse
Freezer warehouse
Freezer warehouse
Freezer warehouse
Processing factory
Processing factory
Commercial outlets
Producer
Commercial outlets
Processing factory
Processing factory
Processing factory
Two fish farms
Three fish farms
Three fish farms
Three fish farms
31 retail markets
Live
Live
Commercial outlets
Gills, skin, guts separately
Skin and belly cavity swabbed
Collar, tail and belly, 25 g
25 g or skin scraping
Skin swabbed
25 g
25 g
Flesh and skin, 25 g
Skin swabbed
Collar, tail and belly, 25 g
Slime layer, 2 g
Skin, 25 g
Flesh under skin, 25 g
Belly-cavity lining, 25 g
Head, 25 g
Tail, 25 g
Trimmings, 25 g
Flesh, 25 g
Skin and surface swabbed
Flesh and skin, 25 g
25 g
Flesh and skin, 25 g
Head, 25 g
Heads, 25 g
Heads, 25 g
Gills, gut, skin 10 g, separately
Flesh, 10 g
Faecal content swabbed
Skin swabbed
Flesh, 25 g
Gills, gut, skin, 10 g, separately
Gills, gut, skin, 10 g, separately
Flesh and skin, 25 g
No. of
samples
10
40
81
215
7
46
50
5
8
61
19
46
22
7
17
9
15
50
48
10
26
22
60
140
117
30
27
45
45
74
30
12
26
% Positive for Listeria
spp. monocytogenes
0
12
2
26
0
65
80
6
0
13
33
54
0
0
21
7
86
4
0
0
88
30
21
65
0
0
47
67
7
8
2
0
15
10
2
4
4
0
15
22
11
51
0
0
0
References
Ben Embarek et al. 1997
Vaz-Velho et al. 1998
Hoffman et al. 2003
Fonnesbech Vogel et al. 2001
Dauphin et al. 2001
Mędrala et al. 2003
Rørvik et al. 1995
Davies et al. 2001
Dauphin et al. 2001
Hoffman et al. 2003
Eklund et al. 1995
Eklund et al. 1995
Eklund et al. 1995
Eklund et al. 1995
Eklund et al. 1995
Eklund et al. 1995
Eklund et al. 1995
Hoffman et al. 2003
Vaz-Velho et al. 1998
Davies et al. 2001
Mędrala et al. 2003
Davies et al. 2001
Autio et al. 1999
Markkula et al. 2005
Markkula et al. 2005
González et al. 1999
Jemmi and Keusch 1994
Jemmi and Keusch 1994
Jemmi and Keusch, 1994
Draughon et al. 1999
González et al. 1999
González et al. 1999
Davies et al. 2001
Table 5 Continued
18
Seafood type (country, area)
Sampling location
Specification
Plaice (UK)
Sardine (Portugal)
Whitefish (USA)
Sablefish1 (USA)
Different species2 (USA)
Different species (USA)
Different species (Denmark)
Different finfish species (India)
Different species (Middle East)
Hake (Argentina)
Mackerel (Argentina)
Blackback (Iran)
Silver carp (Iran)
Different fish species (Japan)
Different fish species (Portugal)
Fish, shellfish, shrimp, etc. (Japan)
Squid (Argentina)
Oysters (USA)
Oysters (USA)
Oysters, mussels, cockles (France)
Shrimps (USA)
Shrimp (All over the world)
Mussel (Argentina)
Crawfish (USA)
Crawfish (USA)
Different shellfish (India)
Shelfish (Middle East)
Commercial outlets
Commercial outlets
Two processing factories
Processing factory
Three processing factories
Two retail markets
Retail markets
Fish market, processing factories
Live
Retail stores
Retail stores
Live
Live
Retail stores
Producers, retail stores
Municipal fish market
Retail stores
Live, collected
Live, collected
Live, collected on shores
Live, collected
Imported to USA, fresh and frozen
Retail stores
Two processing factories
Two processing factories
Fish market, processing factories
Live
Flesh and skin, 25 g
Flesh and skin, 25 g
Flesh, 25 g
Collar, tail, belly, 25g
Collar, belly flap area, 25 g
Fillets, 40 g
25 g
25 g
25 g
25 g
25 g
25 g
25 g
25 g
25 g
10 g
25 g
25 g
25 g
25 g
25 g
25 g
25 g
25 g
25 g
25 g
25 g
1
frozen, 2some frozen
No. of
samples
5
10
67
56
102
320
232
29
40
42
26
28
39
125
25
781
17
35
75
120
74
205
15
78
179
36
15
% Positive for Listeria
spp. monocytogenes
72
37
2
8
29
3
0
55
11
7
27
30
45
44
53
0
0
7
4
9
23
14
17
17
0
0
0
10
2
12
1
18
0
0
9
11
4
0
4
8
12
33
References
Davies et al. 2001
Davies et al. 2001
Hoffman et al. 2003
Hoffman et al. 2003
Norton et al. 2001
Cao et al. 2005
Nørrung et al. 1999
Jeyasekaran et al. 1996
El-Shenawy and El-Shenawy 2006
Laciar and de Centorbi 2002
Laciar and de Centorbi 2002
Basti et al. 2006
Basti et al. 2006
Handa et al. 2005
Mena et al. 2004
Iida et al. 1998
Laciar and de Centorbi 2002
Colburn et al. 1990
Motes 1991
Monfort et al. 1998
Motes 1991
Gecan et al. 1994
Laciar and de Centorbi 2002
Thimothe et al. 2002
Lappi et al. 2004b
Jeyasekaran et al. 1996
El-Shenawy and El-Shenawy 2006
Table 6. L. monocytogenes contamination in fish processing factories and comments on the contamination.
No. of During/before1 % positive for Typing
samples processing L. monocytogenes if used
Sample types
Hot-smoked fish, 1
Raw fish
Fish during processing
Final product
Environment
9
36
15
57
0
3
0
0
Cold-smoked fish, 1
Raw fish
Fish during processing
Final product
Environment
9
36
16
113
44
19
6
32
Hot-smoked fish, 1
Raw fish
Fish during processing
Final product
Environment
9
36
18
76
0
0
0
0
Cold-smoked fish, 1
Raw fish skin
Environment, raw area
Environment, process area
Fish raw area
Fish process area
46
85
23
37
89
19
Product type,
No. of factories
Cold-smoked salmon, 1
Environment, slaughterhouse 83
Environment, smokehouse 147
Fish during processing
71
Final product
65
Cold-smoked salmon,
40
Drains
Fish during process
Cold-smoked and
cold-salted fish, 1
Environment
Raw material
Products
d
d
d
d
d
d
163
55
37
No regular cleaning and disinfection.
Jemmi and Keusch 1994
Jemmi and Keusch 1994
MEE
63
33
15
0
22
References
Jemmi and Keusch 1994
65
33
30
59
71
7
29
23
11
Comments on
L. monocytogenes contamination
PFGE
Primary source of L. monocytogenes
external surface of fish.
Eklund et al. 1995
Final product contamination from
process environment.
Rørvik et al. 1995
Risk factors: rotation of duties,
finding of L. monocytogenes in drains.
Rørvik et al. 1997
Critical contamination sites:
salting and slicing.
One persistent clone for 14 months.
Johansson et al. 1999
Table 6 continued
No. of During/before1 % positive for Typing
samples processing L. monocytogenes if used
Product type,
No. of factories
Sample types
Cold-smoked fish, 1
Raw fish
60
Fish during processing
75
Final product
22
Environment
122
Environment
65
Brine
6
Personnel
19
Air
125
Environment, brine, products 94
b
d
d
d
d
2
29
100
13
30
67
32
0
0
PFGE
Comments on
L. monocytogenes contamination
References
Two major contamination sites
of final products: brining and slicing.
Raw material not important source
of L. monocytogenes of final products.
Some L. monocytogenes clones
predominated.
Eradication program successful.
Autio et al. 1999
Raw fish
Fish during process
Environment
Final products
102
127
206
96
8
18
30
7
Cold-smoked fish, 1
Raw fish
Fish during processing
Final product
Contact surfaces
Environment
18
4
128
50
96
0
0
47
40
17
RAPD
One L. monocytogenes clone persisted
over four years.
Slicing area associated
contamination of final products.
Fonnesbech Vogel et al. 2001
Smoked, cold-smoked,
and gravad fish, 1
Raw fish
Surfaces
Surfaces
Final products
136
281
375
900
11
15
6
6
RAPD
Brining area associated
contamination of final products.
Prevalence may vary significantly
over time at the same factory.
Fonnesbech Vogel et al. 2001
Cold-smoked salmon, 1
Raw salmon
Environment, contact
Environment, no contact
Environment, contact
Environment, no contact
Salmon during processing
Final product
11
71
88
50
80
64
10
PFGE
One dominant and persisted
Dauphin et al. 2001
L. monocytogenes clone that
was not found in raw materials.
Process environment potential
source of final product contamination.
Raw materials not major source of final
product contamination.
20
Cold-smoked fish, 3
18
7
8
4
5
14
21
d
b
d
d
b
b
Ribotyping L. monocytogenes clones persisted in
two factories. Two contamination
sources: raw materials and
environment. All factories had own
L. monocytogenes strains.
Norton et al. 2001
Table 6 continued
No. of During/before1 % positive for Typing
samples processing L. monocytogenes if used
Product type,
No. of factories
Sample types
Cold-smoked fish, 2
Drains
Other environmental sites
Food contact sites
Raw fish
128
96
32
187
d2
d2
d2
d2
63
32
3
4 to 303
Environment
Raw fish
256
128
d2
d2
1
4 to 303
Raw fish
Final product
Environment
Contact surfaces
Employee contact surfaces
Non food surfaces
234
233
553
125
135
162
Shrimp, salmon, cod, 5
Environment
Environment
Floors and drains
Floors and drains
Personnel
Brine
Raw material
Fish during processing
Final product
309
214
91
75
48
23
74
102
104
Cold-smoked salmon, 4
Raw material
86
Brine
14
Final and unfinished products 125
Environment
134
Environment
99
Floors and drains
68
Floors and drains
69
Personnel
48
Smoked fish, 4
21
1
d
d
d
d
b
d
b
d
b
d
b
d
4
1
13
5
10
12
Ribotyping
References
Raw fish and process environment
Hoffman et al. 2003
had different L. monocytogenes
populations. L. monocytogenes
clone persisted over two years.
Factories with similar L. monocytogenes
prevalence values for raw materials, had
disparate values for process contamination.
Ribotyping Environment and cross-contamination Thimothe et al. 2004
the sources of final product contamination.
Raw materials not a major source of L.
monocytogenes of process environment
and final products. Other risk factors
poor employee hygiene and GMP.
10
20
19
27
6
9
14
4
18
16
21
4
3
11
18
25
6
Comments on
L. monocytogenes contamination
PFGE
Cleaning procedures insufficient.
Gudbjörnsdóttir et al. 2004
Cleaning did not eliminate
L. monocytogenes.
Raw materials and processing
environment contamination sources
of L. monocytogenes.
All factories had own
L. monocytogenes flora.
Gudmundsdóttir et al. 2005
d = during and b = before processing, 2 At the beginning of the daily production process, 3 According to different fish species.
2001, Hoffman et al. 2003, Lappi et al. 2004a, Thimothe et al. 2004, Gudmundsdóttir et al.
2005). In addition to persistent L. monocytogenes clones, sporadic L. monocytogenes clones
were also found in the factories (Johansson et al. 1999, Fonnesbech Vogel et al. 2001,
Dauphin et al. 2001, Lappi et al. 2004a, Thimothe et al. 2004). The prevalence of
L. monocytogenes in a certain fish factory varied significantly over time. This was suggested
to be dependent on how busy the period was and the time available for performing the
cleaning and disinfection between the shifts (Fonnesbech Vogel et al. 2001). The processing
environment was found to be the major contamination source for the final products (Rørvik et
al. 1995, Autio et al. 1999, Johansson et al. 1999, Dauphin et al. 2001, Fonnesbech Vogel et
al. 2001, Norton et al. 2001, Lappi et al. 2004a, Thimothe et al. 2004, Gudmundsdóttir et al.
2005). Especially contamination associated with slicing and brining was established (Autio et
al. 1999, Johansson et al. 1999, Dauphin et al. 2001, Fonnesbech Vogel et al. 2001). In
addition potential sources of final product contamination were cross-contamination, job
rotation, employee hygiene and food handling practices (Rørvik et al. 1997, Thimothe et al.
2004). The raw materials have not always been reported as an important final product
contaminant source (Johansson et al. 1999, Dauphin et al. 2001, Thimothe et al. 2004). The
raw materials, however, have clearly been found to be a source of L. monocytogenes
contamination of the final products in certain factories (Eklund et al. 1995, Fonnesbech Vogel
et al. 2001, Norton et al. 2001, Gudmundsdóttir et al. 2005). Another observation was that all the
factories had their own L. monocytogenes contamination patterns and contamination degree at the
process environment and on final products (Dauphin et al. 2001, Hoffman et al. 2003, Lappi et al.
2004a) despite the use of similar raw materials (Thimothe et al. 2004). This was particularly
influenced by the factory design, structure and conditions as well as operational and sanitation
procedures (Hoffman et al. 2003, Thimothe et al. 2004).
2.4.3 Occurrence of Listeria monocytogenes in seafood products
The contamination of seafood products with L. monocytogenes has been widely studied.
Differences occur in the prevalence of L. monocytogenes in different product types as well as
by producers (Table 7). In the case of cold-smoked fish the L. monocytogenes contamination
ranged from 0 to 100%, the mean contamination rate of the sampled products being 30%. The
highest number of L. monocytogenes cells encountered in cold-smoked fish was 2.5x104
CFU/g (Loncarevic et al. 1996). The contamination of hot-smoked fish products, including
those reported only to be smoked, ranged from 0 to 33%. The largest amount of
L. monocytogenes cells found in hot-smoked fish product was 1.3x105 CFU/g (Loncarevic et
22
Table 7. Prevalence of L. monocytogenes and Listeria spp. in seafood products and confirmed highest numbers of L. monocytogenes.
23
Product type
Country
Sampling location
and No.
Cold-smoked salmon
Cold-smoked salmon
Cold-smoked salmon
Cold-smoked salmon
Cold-smoked salmon
Cold-smoked salmon
Cold-smoked salmon
Cold-smoked salmon
Cold-smoked salmon and trout
Cold-smoked salmon and trout
Cold-smoked rainbow trout
Cold-smoked rainbow trout
Cold-smoked halibut
Cold-smoked silver carp
Cold-smoked fish
Cold-smoked fish
Cold-smoked fish
Cold-smoked fish
Cold-smoked fish
Cold-smoked fish
Cold-smoked fish
Cold-smoked fish
Cold-smoked and smoked fish
Cold-smoked fish and shellfish
Hot-smoked rainbow trout
Hot-smoked salmon and whitefish
Hot-smoked whitefish
Hot-smoked fish
Hot-smoked fish
Hot-smoked fish
Hot-smoked fish
Hot-smoked fish and shellfish
Hot-smoked fish
Denmark
Denmark
Denmark
Poland
Italy
France
Norway
USA
Japan
Spain
Finland
Finland
Denmark
Iran
Sweden
Canada
UK, Chile
Switzerland
Switzerland
Finland
Finland
USA
USA
Different countries
Finland
Finland
Finland
Switzerland
Switzerland
Sweden
Finland
Different countries
UK, Ecuador
Producers
Producer
Producer
Producer
Producers, 3
Producer
Producer
Producers, 6
Retail stores
Retail outlets
Producer
Retail outlets
Producers
Retail fish market
Retail markets
Retail outlets
Retail outlets, producers
Import- and export-control
Producer
Retail outlets
Producer
Producers, 3
Producers, 4
Processors, importers
Retail outlets
Retail outlets
Retail outlets
Import- and export-control
Producers, 2
Retail markets
Retail outlets
Processors, importers
Retail outlets, producers
No. of
samples
380
1000
128
44
165
21
65
61
50
54
22
62
40
20
26
116
49
814
16
30
37
96
233
291
42
6
47
471
33
66
48
234
32
% positive for Listeria
spp. monocytogenes
11
6
6
6
6
9
3
13
37
6
47
61
19
10
11
79
24
0
100
15
53
35
12
4
14
6
17
22
11
1
18
2
0
0
12
0
2
2
8
0
Highest number of
L. monocytogenes (CFU/g)
> 1000
> 1100
34
460
> 1000
100 to 1000
25400
132000
References
Jørgensen and Huss 1998
Fonnesbech Vogel et al. 2001
Fonnesbech Vogel et al. 2001
Mędrala et al. 2003
Cortesi et al. 1997
Dauphin et al. 2001
Rørvik et al. 1995
Eklund et al. 1995
Nakamura et al. 2004
González-Rodríguez et al. 2002
Autio et al. 1999
Lyhs et al. 1998
Jørgensen and Huss 1998
Basti et al. 2006
Loncarevic et al. 1996
Dillon et al. 1994
Fuchs and Nicolaides 1994
Jemmi et al. 2002
Jemmi and Keusch 1994
Johansson et al. 1999
Johansson et al. 1999
Norton et al. 2001
Thimothe et al. 2004
Heinitz and Johnson 1998
Lyhs et al. 1998
Lyhs et al. 1998
Lyhs et al. 1998
Jemmi et al. 2002
Jemmi and Keusch 1994
Loncarevic et al. 1996
Johansson et al. 1999
Heinitz and Johnson 1998
Fuchs and Nicolaides 1994
Table 7 continued
24
Product type
Country
Sampling location
and No.
Smoked salmon
Smoked salmon
Smoked salmon
Smoked salmon
Smoked salmon
Smoked trout
Smoked halibut
Smoked fish
Smoked fish
Smoked fish
Smoked RTE fish
Smoked seafoods
Heat-treated seafood
Salmon sauce
Salmon sauce
Processed seafood
Seafood salads
Seafood salads
Seafood salads
Seafood salads
Preserved fish products
Fish fingers
Fish fingers/fish cake
Fish and fish products
Fish butter
Marinated fish
Cold-salted (gravad) fish
Cold-salted fish
Cold-salted fish
Cold-salted fish
Cold-salted rainbow trout
Belgium
Spain
Spain
Norway
Japan
Belgium
Belgium
Iceland
Spain
Canada
USA
USA
Denmark
Italy
Italy
Japan
Belgium
Belgium
Iceland
USA
Denmark
Ireland
Singapore
Italy
Belgium
Switzerland
Sweden
Iceland
Denmark
Finland
Finland
Super markets, 4
Retail outlets
Producers, 8
Producers
Retail stores
Super markets, 4
Super markets, 4
Retails stores, producers
Retail outlets
Retail outlets
Producers, 4
Retail markets
Producers
Producer
Producer
Fish markets
Super markets, 4
Supermarket chain
Retails stores, producers
Retail markets
Retail outlets
Retail outlets, producers
Retail outlets, producers
Super markets, 4
Import- and export-control
Retail markets
Retails stores, producers
Producers
Retail outlets
Retail outlets
No. of
samples
42
52
100
33
92
15
18
31
170
142
519
2644
79
32
42
247
45
362
37
2446
335
20
16
3160
3
125
58
23
176
32
43
% positive for Listeria
spp. monocytogenes
19
38
41
0
33
35
19
27
28
9
5
0
33
3
22
Highest number of
L. monocytogenes (CFU/g)
< 10
>100
25
8
49
32
95
0
61
4
13
6
0
5
27
27
16
5
11
15
19
6
0
38
21
22
29
50
33
> 100000
> 1000
100 to 1000
> 100
1000 to 10000
3400
> 1000
References
van Coillie et al. 2004
Aguado et al. 2001
Vitas et al. 2004
Rørvik and Yndestad 1991
Inoue et al. 2000
van Coillie et al. 2004
van Coillie et al. 2004
Hartemink and Georgsson 1991
Dominguez et al. 2001
Dillon et al. 1994
Lappi et al. 2004a
Gombas et al. 2003
Jørgensen and Huss 1998
Pourshaban et al. 2000
Pourshaban et al. 2000
Iida et al. 1998
van Coillie et al. 2004
Uyttendaele et al. 1999
Hartemink and Georgsson 1991
Gombas et al. 2003
Nørrung et al. 1999
Sheridan et al. 1994
Ng and Seah 1995
Busani et al. 2005
van Coillie et al. 2004
Jemmi et al. 2002
Loncarevic et al. 1996
Hartemink and Georgsson 1991
Jørgensen and Huss 1998
Johansson et al. 1999
Lyhs et al. 1998
Table 7 continued
25
Product type
Country
Sampling location
and No.
No. of
samples
Cured seafood
Shrimp
Shrimp
Shrimp
Shrimp
Shellfish and shrimp
Shellfish
Shellfish
Shellfish
Shellfish
Crawfish
Crawfish
Crabmeat
Crab
Crabmeat/scallops
Crustaceans
Surimi
Minced fish
Roe
RTE products
RTE-salmon
RTE-seafood
Raw RTE-seafood
Denmark
Iceland
Norway
Different countries
Different countries
Iceland
Portugal
Italy
Chile
Japan
USA
USA
USA
USA, China
Singapore
Italy
Different countries
Norway
Japan
Switzerland
Different countries
Nordic Countries
Japan
Producers
91
Producers, 26
3331
Producers
16
Wholesales
49
Retail outlets
20
Retails stores, producers
22
Producers, retail stores
8
Retail outlets, producers
1494
Producers, markets, restaurants 268
Retail stores
16
Producers
78
Retail stores
31
Producers
126
Wholesales
7
Retail outlets, producers
16
Retail outlets, producers
347
Wholesales
25
Producers
8
Retail stores
67
Import- and export-control
151
Wholesales
32
Producers
63
Retail stores
213
% positive for Listeria
spp. monocytogenes
8
5
0
10
5
4
2
18
8
20
5
0
0
12
0
0
3
8
14
0
0
0
12
10
7
31
5
3
Highest number of
L. monocytogenes (CFU/g)
< 10
> 100
References
Jørgensen and Huss 1998
Valdimarsson et al. 1998
Rørvik and Yndestad 1991
Farber 1991
Farber 1991
Hartemink and Georgsson 1991
Mena et al. 2004
Busani et al. 2005
Cordano and Rocourt 2001
Handa et al. 2005
Thimothe et al. 2002
McCarthy 1997
Rawles et al. 1995
Farber 1991
Ng and Seah 1995
Busani et al. 2005
Farber 1991
Rørvik and Yndestad 1991
Handa et al. 2005
Jemmi et al. 2002
Farber 1991
Gudbjörnsdóttir et al. 2004
Inoue et al. 2000
al. 1996). Seafood salad L. monocytogenes contamination occurred at a rate between 4.7 to 27%
and the highest number of cells found was 100-1000 CFU/g (Gombas et al. 2003). The
contamination of cold-salted fish varied between 21 to 50% and the largest number of
L. monocytogenes cells found was 3.4x103 CFU/g (Loncarevic et al. 1996). In Japan,
contamination occurred in 10% (7/67) of the studied roe samples (Handa et al. 2005). The
overall L. monocytogenes contamination rate of seafood products was 7.8% according to Table 7.
The amounts of L. monocytogenes found in different seafood products that have been
suspected of causing listeriosis cases vary. In Finland, a listeriosis outbreak was connected
with the consumption of cold-smoked rainbow trout. The cold-smoked rainbow trout from the
same lot and the same retail store was found to contain 1.9x105 CFU/g of L. monocytogenes
(Miettinen et al. 1999). In a case with smoked mussels, L. monocytogenes amounts of 1.6x107
CFU/g and 3.2x106 CFU/g occurred in the mussels from the patients’ refrigerators (Mitchell
1991). High numbers of L. monocytogenes (2.1x109 CFU/g) were also found in imitation crab
meat that caused a listeriosis outbreak (Farber et al. 2000). On the other hand in a listeriosis
outbreak associated with the consumption of cold-salted rainbow trout the detected amounts
of L. monocytogenes in the fish from two patients’ refrigerators were low (<100 CFU/g, 6200
CFU/g) (Ericsson et al. 1997).
2.5 Control of Listeria monocytogenes in fish processing
The policy regarding the presence and acceptable amounts of L. monocytogenes in different
seafood products varies in different countries, from zero tolerance (e.g. in the USA) to an
acceptable amount of less than 100 CFU/g that has been estimated as a level when the risk of
listeriosis is very low even for high-risk groups (Farber et al. 1996, Buchanan et al. 1997). In
Finland, the Finnish food safety authority also has a guideline for the control of
L. monocytogenes. The level of L. monocytogenes in seafood products at the time of
consumption should be less than 100 CFU/g. When the product leaves the production plant
L. monocytogenes should not be detectable. In addition there are instructions like additional
samplings or product recalls, for situations when L. monocytogenes or Listeria spp. are found
in products or from plant environment.
To efficiently control L. monocytogenes, every potential route of entry and cross
contamination must be monitored (Gravani 1999) plus good manufacturing practices need to
be followed (Blanchfield 2005). The production facilities are important, as plants in which
production facilities were in a good state of repair had a lower risk of L. monocytogenes
26
contamination than those with moderate or heavy wear and tear (Rørvik et al. 1997). In
addition, the facilities should be designed or arranged to restrict or eliminate the transmission
of people, equipment and conveyance between raw, processing, packaging and shipping areas
as the cross contamination between raw and finished product areas is a major source of
contamination (Lappi et al. 2004a). Well-designed lines and facilities enable good working
routines and effective departmentalisation of facilities help in controlling both employees and
product traffic (Autio et al. 2004). The employee movement between departments, due to
rotation of the assigned duties, was found to be a risk factor for L. monocytogenes
contamination of final products, especially if limited or no precautions were taken to avoid
spread of bacteria (Rørvik et al. 1997). Cross contamination by employees and the
environment of the processing plants may represent important causes of contamination of the
finished product (Thimothe et al. 2004). Separate equipment, including tools employed by
maintenance persons, should be available for use in raw and finished product areas (Gravani
1999). In addition all equipment within the factory should be designed to minimise cross
contamination to products. Certain points of the processing chain, such as the machines, are
particularly susceptible to contamination, because of the difficulties in efficient cleaning
(Dauphin et al. 2001). The slicing and brining processes were the most critical steps of the
fish production line mainly due to difficulties with cleaning the equipment thoroughly (Autio
et al. 1999, Johansson et al. 1999). In addition, L. monocytogenes was extremely difficult to
eliminate using routine disinfection procedures in a meat-bone separator which is complex
equipment (Lappi et al. 2004a). The prevention of L. monocytogenes contamination in
products is based on avoiding the colonisation of processing environment and equipment with
L. monocytogenes. The hygienic aspects should be stressed when selecting new processing
equipment as the complex construction of equipment often hampers the cleaning and
disinfection practices that cannot be applied due to the effects on equipment materials (Autio et
al. 2004).
Listeria spp., including L. monocytogenes, have been most frequently isolated from floor
drains and floors, thus suggesting that these areas may function as reservoirs for Listeria in
food processing facilities (Rørvik et al. 1997, Norton et al. 2001, Thimothe et al. 2004). These
should be thoroughly cleansed and disinfected daily but high-pressure hoses should never be
used, since such practices readily promote the spread of Listeria to nearby equipment and
other areas of the factory through splashing and the generation of aerosols (Gravani 1999).
Clean and especially dry floors are important for the control of Listeria in processing plants
(Thimothe et al. 2004).
27
Disinfection is the final step in eliminating L. monocytogenes and other food borne pathogens
as well as the myriad of spoilage organisms present in the production environment. Since the
presence of organic debris readily decreases the effectiveness of disinfecting agents against
L. monocytogenes (Best et al. 1990), it is important to remember that every item must first be
thoroughly cleaned before it is disinfected. L. monocytogenes is sensitive to disinfectants
commonly employed in the food industry. Disinfection with hot water is not advised, since
sufficiently high water temperature can not be easily maintained (Gravani 1999). In some
cases, however, the use of hot water (80 °C), heating in oven (80 °C) and treating with gas
flame or a hot steam treatment has proven efficient in eradication of L. monocytogenes in a
fish factory (Autio et al. 1999, Lappi et al. 2004a). In addition to inadequate separation
between raw and finished product resulting from faulty factory design, indifferent attitudes of
employees toward proper cleaning and disinfecting has been most frequently cited as factors
that promote post processing contamination. Effective cleaning and disinfection programs for
standard operating procedures for every factory job along with master schedules listing the
frequency of cleaning and disinfection procedures are needed (Gravani 1999). The
effectiveness of the cleaning and disinfection programs should be verified through daily
microbiological analysis of both product and environmental samples gathered from all areas
of facility. During environmental sampling, the efficacy of cleaning and disinfection
procedures can be easily determined through the use of ATP bioluminescence monitoring
systems. Routine testing of environmental samples for Listeria spp. remains, however, a critical
component of any disinfection verification program (Gravani 1999). Development of focused
clean-up and disinfection procedures as well as implementation of the HACCP programme
are of great importance in the prevention of L. monocytogenes colonisation (Autio et al. 2004).
Consistent monitoring of L. monocytogenes contamination over time should be a component
of every L. monocytogenes control strategy (Hoffman et al. 2003). The prevalence data and
subtyping of L. monocytogenes provide a base for implementing effective cleaning and
disinfecting procedures focusing on L. monocytogenes niches and transmission pathways
(Thimothe et al. 2004). A plant-specific Listeria control program should also include
strategies to minimise both the raw material and the environmental contaminations,
procedures to prevent cross-contamination and employee training (Lappi et al. 2004a). Even
when handled under the best possible conditions, raw seafood or processing environment will
probably never be completely free from L. monocytogenes (Gravani 1999, Fonnesbech Vogel
et al. 2001, Autio et al. 2004).
28
3 AIMS OF THE STUDY
The aims of the present thesis were to investigate the prevalence and sources of
L. monocytogenes in different stages of the fish production chain and to improve the safety of
rainbow trout roe products. The specific aims were as follows:
1.
to determine the prevalence and location of L. monocytogenes in farmed rainbow trout
(I),
2.
to determine the prevalence of L. monocytogenes and the quality of Finnish roe products
in the retail level (II),
3.
to ensure the safety of rainbow trout roe in regards to L. monocytogenes elimination and
quality maintenance of pasteurisated, refrigerator stored roe (III),
4.
to study the presence of L. monocytogenes and Listeria spp. on fish factory surfaces and
in final fish products as well as the association of surface hygiene and occurrence of
Listeria spp. (IV),
5.
to investigate the sources and ecology of L. monocytogenes and Listeria spp. in fish
farming (V) and
6.
to study the distribution of L. monocytogenes isolates from raw fish, fish production
factories and the final fish products typed with pulsed-field gel electrophoresis (V).
29
4 MATERIALS AND METHODS
4.1 Sampling of raw material rainbow trout (I)
A total of 510 rainbow trout in lots of 10 to 50 fish each originating from fish farms in Finnish
lakes (four lots) and sea areas (14 lots) were studied. The fish were individually packed into
plastic bags straight after slaughter, and kept on ice. In addition to full-grown fish, two
juvenile rainbow trout lots of 50 fish each from different farms were investigated. Three
separate samples from each full-grown fish were taken: 1) the fish gills, with gill arches, were
aseptically removed, 2) the peritoneal cavity was opened and the viscera without heart and
kidneys were removed aseptically and 3) the fish skin was peeled off. The gill, viscera and
skin samples were each thoroughly chopped using scissors. After chopping, pooled samples
were formed separately from gill, viscera, and skin samples of five fish, resulting in three
samples altogether. Only part of these original samples were taken into the pooled sample, and
the rest of the original samples were incubated for five days at 5 °C, after which the samples
were frozen. All original samples in the pool were analysed later if the pooled sample was
found to be Listeria spp. positive. Two juvenile fish lots were chopped without separation of
sample locations and pooled, each pool containing five fish.
4.2 Rainbow trout roe and its pasteurisation (II, III)
4.2.1 Retail level and fresh roe (II, III)
A total of 141 fresh, frozen or frozen-thawed roe samples from Finnish rainbow trout
(Oncorhynchus mykiss), whitefish (Coregonus lavaretus) and vendace (Coregonus albula)
were bought. The roe samples were bought from 46 different retail stores, shops and markets
of various sizes in eight towns, and they originated from 26 different factories and suppliers in
Finland. Frozen roe samples were allowed to thaw at 5.0 °C for 18-24 h before analysis. From
at least 30 roe samples of each fish species (total 92) the amount of Listeria spp., aerobic and
coliform bacteria as well as sensory quality were analysed and an additional 49 roe samples
were analysed for Listeria spp. presence.
Fresh Finnish rainbow trout (Oncorhynchus mykiss) roe, used in study III was obtained from a
wholesaler where it was cleaned, salted (2.5% wt/wt), and vacuum packed one to two days
before further studies and processing in the laboratory.
30
4.2.2 Listeria monocytogenes strains (III)
Study III used four L. monocytogenes strains isolated during study II from rainbow trout,
whitefish and vendace roes. The strains were adapted to 10 °C by incubating them separately
three times at 10 °C for five days in Trypticase Soy Broth (Becton, Dickinson and Company,
Sparks, MD, USA).
4.2.3 D- and z-value determination (III)
Determination of D-values for a mixture of equivalent amounts of four L. monocytogenes
strains in rainbow trout roe was performed twice at both test temperatures (60 °C and 63 °C).
Cold adapted L. monocytogenes strains were inoculated into fresh rainbow trout roe and
mixed well resulting in a L. monocytogenes concentration of around 5x105 CFU/g. The roe
was incubated for five days at 10 °C so the cells achieved the stationary growth phase and
were the most resistant to heat treatment. The amount of L. monocytogenes cells in roe was
108 CFU/g before heat treatment. The inoculated roe was packaged into heat sealed plastic
film pouches (6 cm x 6 cm x 0.4 cm). Each pouch contained 15 g of roe. The heat treatment
was performed in a water bath (WB 29, Memmert, Schwabach, Germany) and the
temperature was measured inside a control roe pouch not inoculated with L. monocytogenes
and in a water bath with temperature sensors (K-type thermo element, Fluke 52 II, Fluke
Corp., Everett, WA, USA). The stability of the water temperature during the experiment was
± 0.2 °C. The test was initiated when the control pouch reached the desired temperature
(60 °C or 63 °C). Triplicate samples were taken out of the water bath each time (30 s to 150 s
intervals) and put into an ice-water mixture. The amount of L. monocytogenes in the pouches
was determined by cultivation on the same day. Linear regression analysis of the thermal
destruction of L. monocytogenes cells was performed in each heat treatment experiment. The
D-value was calculated as a negative reciprocal of the mean of duplicate slopes at both 60 °C
and 63 °C.
The z-value is the temperature difference required to yield a 10-fold change in the heating
time and still destroy 90% of the L. monocytogenes cells. The z-value was calculated using
the equation z = (T2-T1) / log(D1/D2), where D1 is the time needed to destroy 90% of
L. monocytogenes cells at temperature T1, and D2 is the time needed to destroy 90% of the
L. monocytogenes cells at temperature T2.
31
4.2.4 Pasteurisation of rainbow trout roe (III)
Roe was packaged into glass jars (100 g) and vacuum sealed (Mini-VacuumTwister, MVT34/3, Rainer Naroska engineering, Bad Salzuflen, Germany). Pasteurisation of the roe was
performed in an autoclave (Stock Pilot Rotor 900, Herman Stock Maschinenfabrik,
Neumünster, Germany) as a water spray circulation run. Four temperature sensors (T type
thermo element, Cu/CuNi, Ellab A/S, Roedovre, Denmark) installed inside four glass jars
situated in different positions in the autoclave monitored the run temperature. One sensor
monitored the temperature of the water spray. Temperatures were recorded with the Dasylab
(Dasytec, Amherst, NH, USA) measurement program. Pasteurised roe jars were cooled with
water spray circulation (7 °C) in the autoclave after pasteurisation.
Two experiments were conducted to evaluate the shelf-life of the pasteurised roe. Fresh
vacuum packaged roe was pasteurised for 10 min at 65 °C and 15 min at 62 °C. Control
samples were frozen (-18 °C) from the same fresh roe lot, but without pasteurisation. After
pasteurisation the jars of roe were stored at 3 °C for 31 weeks and the sensory and microbial
quality were analysed five and four times, respectively. Further microbiological analysis was
performed on five jars of each roe type after the storage time had elapsed.
Two pasteurisation experiments with L. monocytogenes -inoculated roe were performed
(10 min at 62 °C and at 65 °C). Before pasteurisations the roe was similarly inoculated and
incubated as the roe in the D-value experiments. After pasteurisation the jars were stored for
two weeks at 10 °C and L. monocytogenes was qualitatively analysed.
4.2.5 Pasteurisation values (III)
Pasteurisation values for pasteurisation experiments were calculated using the equation
P
(T −T ) / z
= 10 ref (Stumbo, 1973), where P is the pasteurisation value (min), td is the heat death
td
time (min), Tref is the reference temperature, and z-value was already determined with the
help of D-values. Because the reference temperature should be lower than the actual
pasteurisation temperature Tref value of 60 °C was chosen. The pasteurisation values were
calculated on a spreadsheet program (Microsoft Excel 2002, Microsoft Corporation,
Redmond, WA, USA) at 30 s intervals and added together. Calculation was started when the
last temperature sensor inside the roe jar reached 50 °C and ended as it dropped below 50 °C.
32
4.3 Sampling in a fish farm (I, V)
A fish farm that had severe L. monocytogenes contamination in fish according the study I was
further sampled 14 times. The fish cleaning house was sampled (17) during the first sampling
occasion. The fish farm environment samples included fish (78), farming nets (17), equipment
(5), water (78) and sea bottom soil (43) samples. Fish were analysed as pooled samples of gills
from one or two fish (25 g). Water samples were directly analysed from 25 ml and bigger
volumes (three or four litre) were first prefiltered and then sterile filtered. Surface samples
were taken with sterile cotton gauzes, the gauzes being moistened with sterile peptone water if
necessary. Surface areas of at least 100 cm² were sampled. The sea bottom soil samples (25 g)
were mainly taken inshore, except for seven samples that were taken from five to 20 m deep
sea areas. The fish farming nets and facilities were situated near (40 m) the shore. A nearby
(10 km) meteorological measurement site measured the mean monthly rainfall (1970 to 2000)
and rainfall from 2001 to 2004.
4.4 Sampling in fish processing factories (IV)
A total of 943 surface samples were taken for Listeria spp. analyses from 23 factories.
Hygiene samples from surfaces (866) with four other methods (Table 8) were taken from 28
factories. Surface samples were taken in the morning after washing and cleaning just before
the start of a normal workday. Most of the samples, on average 30-40 samples from each
factory, were taken from surfaces considered important for process hygiene, e.g. knives,
chopping boards, conveyor belts, control panels and aprons. Some samples for Listeria spp.
analyses, five to seven per factory, were also taken from floors, drains and other lower hygiene
areas. In addition, two to six samples of raw, marinated, cold-salted, cold-smoked or smoked
fish or roe products were taken from each factory to be analysed for Listeria spp. presence.
4.5 Microbial analyses
4.5.1 Isolation and identification of Listeria spp. and Listeria monocytogenes (I, II, III, IV, V)
Listeria spp. were analysed according to the ISO method (Anonymous 1996) for detection
with a two-step enrichment with half-Fraser (Half Fraser broth, Oxoid, Basingstoke, UK;
30 °C, 1d) and Fraser (Fraser broth, Oxoid; 37 °C, 2d) enrichment broths (studies I, II, III, V)
or according to Nordic Committee on Food Analysis method (Anonymous 1999) using onestep enrichment with EB-broth (Listeria Enrichment broth, Oxoid; 30 °C, 2d; study IV). The
selective plates used were Oxford (Oxoid; 37 °C, 2d; studies I, II, III, IV, V), LMBA
(Tammer-Tutkan Maljat, Tampere, Finland; 37 °C, 2d; studies I, II, III, V) and PALCAM
33
(Oxoid; 37 °C, 2d; study IV). Sample size was 25 g or 25 ml. The filter and surface gauze
samples were weighed and incubated in enrichment broth in the same ratio as the 25 g
samples. Listeria from the factory surfaces (study IV) was sampled using contact plates
(25.5 cm2) of Oxford-agar (Oxoid). The contact plates were incubated for two days at 37 °C.
Gram-staining, catalase test, haemolysis testing (Orion Diagnostica, Espoo Finland or
bioMérieux, Marcy l'Etoile, France, 37 °C, 2 d) and API-Listeria test (bioMérieux) were
performed for suspected Listeria spp. colonies instead of testing the carbohydrate utilization
and the CAMP test.
4.5.2 Enumeration of Listeria spp. (II, III)
Performance of Listeria spp. enumeration (study II) was according to ISO method for
enumeration with 25 g samples on PALCAM (Oxoid; 37 °C, 2d) plates (Anonymous 1996).
At least five suspected Listeria spp. colonies per plate were identified as mentioned above.
Direct plating of roe samples in the D-value determination study (III) was performed from
10 g samples on Oxford agar plates not supplemented with selective components. Plates were
incubated for two days at 37 °C.
4.5.3 Analysis of aerobic, anaerobic, coliform and enterobacteria, fungi and ATP (II, III, IV)
The microbial analysis of retail roe samples and surface hygiene samples were performed
according to Table 8.
Table 8. Microbial methods used in analysis of aerobic, anaerobic and coliform bacteria and fungi in
roe samples and the methods used for surface hygiene samples in fish factories.
Analysis
Sample
Method
Plate/kit
Aerobic bacteria
Coliform bacteria
Fungi
Anaerobic bacteria
Anaerobic bacteria
Anaerobic bacteria
Aerobic bacteria
Enterobacteria
Fungi
ATP
10 g, roe
10 g, roe
10 g, roe
10g, roe
10g, roe
10g, roe
Surface
Surface
Surface
Surface
Spread-plating
Pour-plating
Spread-plating
Spread-plating
Spread-plating
Spread-plating
Contact agar
Contact agar
Contact agar
Swabbing
Plate count agar (Difco Sparks, MD, USA) 30 °C, 3d
Violet red bile agar (Difco)
37 °C, 2d
Potato dextrose agar1 (Difco)
25 °C, 5d
Reduced plate count agar (Difco)
10 °C, 10d2
Reduced plate count agar (Difco)
30 °C, 3d2
Reinforced clostridial medium (Difco)
37 °C, 7d2
®
Hygicult TPC (Orion Diagnostica)
30 °C, 2d
Hygicult® E (Orion Diagnostica)
37 °C, 2d
Hygicult® Y&F (Orion Diagnostica)
25 °C, 5d
Surface monitoring kit (Bio-Orbit, Turku, Finland)
1
Incubation
Supplemented with 0.4% chloramphenicol (Sigma Chemical, St. Louis, MO, USA), 0.4% chlortetracycline
(Sigma) and 0.02% Triton-X (Fluka Chemie, Buchs, Germany)
2
Incubation in anaerobic jars (Anoxomat WS8000, Mart® Microbiology, Lichtenvoorde, Netherlands)
34
4.5.4 Identification of bacteria from pasteurised roe (III)
Identification of isolated bacteria from pasteurised roe samples was performed after Gramstaining, oxidase, catalase, and indole tests with API50 CHB (bioMérieux) or Chrystal ID
(Becton, Dickinson and Company) according the manufacturer’s instructions.
4.6 Sensory analysis (II, III)
A modified general descriptive profile method (study II, Anonymous 1985) and a descriptive
sensory analysis method, conventional profiling (study III, Anonymous 2003) were used. In
study II the panel scored four attributes: appearance as dryness (freeness from visible liquid),
odour freshness, texture as egg firmness and freshness of taste on a continuous intensity line
scale from zero (low intensity) to ten (high intensity). The panellists were also allowed to give
verbal descriptions of the samples and add comments. In study III the following eight
attributes were evaluated: freshness of odour and taste, taste intensity, brightness of colour,
amount of free liquid, intactness and firmness of eggs and total quality. The attribute
intensities were rated on a continuous unstructured graphical line scale from zero (low
intensity) to five (high intensity).
4.7 Typing of Listeria monocytogenes isolates
4.7.1 Ribotyping (I)
Ribotyping was performed for 14 L. monocytogenes positive samples including water, surface
and fish samples from the first sampling occasion in the studied fish farm. Ribotyping was
carried out for two isolates per sample, altogether 28 isolates. Performance of ribotyping
followed the manufacturer’s standard instructions, as described by Bruce (1996). DNA was
digested with EcoRI (DuPont Qualicon, Wilmington, Del., USA). The automated system
processed the batches and generated a pattern for each sample and a marker lane using
proprietary algorithms. Isolates were assigned to a ribogroup from the database, or a new one
was created and similarities were calculated (Qualicon software v. 12.2). Definition of a
ribogroup was a set of closely related patterns (threshold similarity ≥ 0.97) that were
mathematically indistinguishable from one another by the system.
4.7.2 PFGE-typing (V)
Fish farm ecology and the relationships of L. monocytogenes isolates between farmed fish,
fish processing factories and fish products were studied with PFGE-typing. Before subtyping
with PFGE 98 L. monocytogenes isolates from the fish farm samples (study V), 47
35
L. monocytogenes isolates from raw rainbow trout (study I) and 36 L. monocytogenes isolates
from nine fish processing factories collected earlier partly in study III were frozen stored.
DNA was isolated according to the PulseNet 4-h protocol (Graves and Swaminathan 2001)
with minor modifications. In brief, the cell density of overnight grown bacteria was adjusted
to 0.8 in TE-buffer (0.01 M Tris [Sigma], 0.01 M EDTA [Riedel-de Haën, Seelze, Germany],
pH 8.0). Cell suspensions with 10 mg/ml lysozyme (Sigma), molten InCert Agarose
(BioWhittaker Molecular Applications, Rockland, ME, USA), 1% sodium dodecyl sulphate
(Sigma) and 0.2 mg/ml Proteinase K (Roche Molecular Biochemicals, Mannheim, Germany)
were dispensed into plug molds (Bio-Rad Laboratories, Hercules, CA, USA). The agarose
plugs were lysed in lysis buffer (0.05 M Tris, 0.05 M EDTA, 1% sodium lauryl sarcosine
[Sigma], 0.15 mg/l Proteinase K) for 2 h at 53 °C in a water bath shaker. After proteolysis, the
lysis solution was removed and the plugs were washed. Plugs were digested with AscI (New
England BioLabs, Beverly, MA, USA) in buffer solutions according to the manufacturer's
instructions. DNA fragments were electrophoretically separated through 1% agarose gel
(Pulsed Field Certified Agarose, Bio-Rad Laboratories) in 0.5 x TBE buffer (Bio-Rad
Laboratories) at 200 V, 14 °C in CHEF Mapper® XA PFGE apparatus (Bio-Rad
Laboratories). The pulse times ramped from 1 to 35 s for 18 h. Lambda Ladder PFG marker
(New England BioLabs) was used for fragment size determination. After electrophoresis, the
gels were stained with ethidium bromide and subsequently photographed (Gel Doc 2000
system, Bio-Rad Laboratories). The resulting pulsed-field electrophoretic macrorestriction
patterns were compared using Bionumerics software (Applied Maths, Sint-Martens-Latem,
Belgium). A dice coefficient (position tolerance 1.0%) expressed different PFGE pattern
similarity. The clustering and construction of dendograms were performed by the unweighted
pair group method using arithmetic averages (UPGMA).
4.8 Statistical analyses (I, II)
Listeria spp. and L. monocytogenes prevalence values for different locations in fish were
compared using Pearson's chi-square tests (I). Differences in the microbial and sensory
quality of roe between the fish types and storage conditions were analysed with 3x3 multiand univariate analyses of variances (II). In addition, one-way multivariate analyses of
variances were performed separately for each fish type and storage condition. Tukey's post
hoc test was used to determine which of the fish types and storage conditions differed from
each other. Pearson product-moment correlations were calculated to analyse relationships
between the sensory and microbiological measures (II).
36
5 RESULTS
5.1 Prevalence and location of Listeria monocytogenes in farmed rainbow trout (I)
Two-thirds of the fish lots (11/18) contained Listeria spp. and more than one-fourth contained
L. monocytogenes (5/18). Of the pooled fish samples, 15% (15/103) were contaminated with
L. monocytogenes and 35% (36/103) with Listeria spp. Contamination of individual fish,
studied after frozen storage, was lesser (Table 9). The freezing destroyed part of the Listeria
spp. population in fish samples since seven pooled samples positive for Listeria spp. were not
found to contain Listeria spp. in any of the individual fish samples after the frozen storage.
Listeria spp. occurred most often in the fish gill samples (Table 9), only a few times in skin
samples and once in viscera samples. Almost all identified L. monocytogenes isolates were
found in gills, while only one in viscera and one in skin samples. Many gill samples within
the lot with the L. monocytogenes positive skin sample were also contaminated with
L. monocytogenes. In the case of the L. monocytogenes positive viscera sample, the viscera
were the only sample containing L. monocytogenes.
Table 9. Prevalence of Listeria spp. and L. monocytogenes in individual fish according to location.
Percentages given in brackets refer to the division of number of positive samples by the total number
(510).
Sample
Listeria spp.
No. (%)
Gills
Skin
Viscera
82 (16)a
14 (2.7)b
1 (0.2)b
43 (8.4)a
1 (0.2)b
1 (0.2)b
Total fish
91 (18)
44 (8.6)
L. monocytogenes
No. (%)
ab
Different superscripts within a column refer to statistically significant (p < 0.0001) difference in
prevalence.
Clear differences occurred in the Listeria spp. and L. monocytogenes prevalences among the
fish farms. All or most of the pooled fish samples at seven farms were contaminated with
L. monocytogenes or other Listeria spp., at four farms contamination of fish lots was only
sporadic and at seven farms no Listeria spp. appeared in any fish. No association emerged
between certain sea or lake areas and Listeria spp. contamination. All sampled areas
contained fish farms with and without Listeria spp. contamination. No Listeria spp. was found
in the juvenile fish studied.
37
5.2 Safety and quality of Finnish retail level roe (II)
5.2.1 Prevalence of Listeria monocytogenes (II)
Nearly a fifth of roe samples were contaminated with Listeria spp. and a minor amount with
L. monocytogenes (5%). The prevalence of L. monocytogenes and Listeria spp. (p < 0.01)
differed significantly between storage conditions (Table 10) fresh-bought roe products
containing more often L. monocytogenes than fresh and frozen-thawed roes. Listeria spp.
occurred more often in rainbow trout roe (p < 0.001) than in roes of other fish species (Table
10). The interactions between fish species and storage condition were not significant
(p = 0.60). Listeria spp. quantities found with direct plating were normally low. One fresh
rainbow trout roe sample contained L. monocytogenes at a level of 60 CFU/g. L. innocua
occurred in three fresh rainbow trout roe samples and one fresh whitefish roe sample with
greater than 10 but less than 100 CFU/g. Most of the Listeria positive samples resulted from
enrichment procedures. Listeria spp. or L. monocytogenes occurrences did not depend on the
producer, since all the L. monocytogenes positive roes were processed by different producers
and 25 Listeria spp. positive roes originated from 18/26 different producers.
Table 10. Prevalence of Listeria spp. and L. monocytogenes in different roe product types.
Roe storage condition/
fish species of roe
No. of
samples
Positive for
Listeria spp. (%)
Positive for
L. monocytogenes (%)
Frozen
Frozen-thawed
Fresh
59
48
34
5 (8.5)b
5 (10)b
15 (44)a
0 (0)a
1 (2.1)a
6 (18)b
Rainbow trout
Whitefish
Vendace
50
45
46
18 (36)a
5 (11)b
2 (4.3)b
4 (8.0)
2 (4.4)
1 (2.2)
141
25 (18)
7 (5.0)
Total roe samples
ab
Different superscripts within storage types (p < 0.01), fish species (p < 0.001) and columns refer to
statistically significant difference in prevalence.
5.2.2 Microbial and sensory quality (II)
The mean count and median of total aerobic bacteria of all roe samples was 6.6 ± 1.5
logCFU/g and 7.1 logCFU/g, respectively. Bacterial counts of vendace roe were significantly
(p < 0.05) higher than counts in rainbow trout roe, but not significantly different from those of
whitefish. This difference between fish species was highest when the roes were bought fresh
(p < 0.05), although a similar tendency was also observed in frozen roe. With regard to
individual fish species and storage conditions, the contamination was significantly higher
38
(p < 0.05) in fresh vendace roe samples than in those of frozen rainbow trout and whitefish
roes.
The mean count and median of coliform bacteria of all roe samples was 3.2 ± 1.7 logCFU/g
and 3.4 logCFU/g, respectively. There were no significant differences among fish species, but
significantly (p < 0.05) fewer coliform bacteria occurred in frozen roe than in fresh roe
samples. In addition, significantly (p < 0.05) more coliform bacteria were found in fresh
vendace roe than in either frozen vendace or frozen rainbow trout roes.
Multivariate tests showed significant differences in the sensory scores between the storage
conditions (p < 0.001), fish species (p < 0.001) and interactions between the two (p < 0.01).
The ratings of all sensory characteristics correlated highly with each other. With respect to
taste and odour freshness the roe quality was best in rainbow trout, however, appearance and
structure evaluation was best in vendace roe samples. Roe samples bought fresh had on
average a fresher odour and taste than frozen and frozen-thawed roe samples, seen in all three
fish species. Sensory defects in roe samples were described as rancid, musty, fishy, metallic,
yeast- and mould-like, slimy, gruel-like and eggs having hard skin. There was no significant
correlation between results obtained by sensory and microbial methods.
5.3 Pasteurisation of rainbow trout roe (III)
5.3.1 D- and z-values of four Listeria monocytogenes strain mixture in rainbow trout roe (III)
Heat treatments of the mixture of the four L. monocytogenes strains resulted in D-values of
1.60 min and 0.44 min at 60 °C and at 63 °C, respectively, and the z-value calculation was
5.36 °C. In both temperatures the linear regression lines of the thermal death of
L. monocytogenes cells were parallel with the duplicate experiment at the same temperature.
5.3.2 Microbial quality (III)
Microbial quality of pasteurised roe was excellent during the entire 31 week storage time as
the aerobic, coliform and fungal counts were below detection levels. Frozen control roes were
also of good microbial quality and the level of aerobic and coliform bacteria as well as fungi
were low, mostly under 103 CFU/g. No anaerobic psychrotrophic bacteria were detected in
any of the roes nor were there aerobic psychrotrophic bacteria in the pasteurised roes at the
end of the storage time. The level of aerobic psychrotrophic bacteria in control roes were
below the level of aerobic mesophilic bacteria that was detected in the same roes. One
39
pasteurised roe (1/10) and one control roe (1/5) showed growth of a few anaerobic mesophilic
colonies on plate count agar. Bacillus circulans was identified from the pasteurised (62 °C)
roe sample. From three pasteurised roe (3/8) and one control roe sample (1/5) a few colonies
growing on clostridium medium were found. As these colonies, however, could not be
identified with Chrystal ID they were not C. botulinum nor C. perfringens.
5.3.3 Sensory quality (III)
The quality of both the pasteurised and frozen control roe samples were good until 24 weeks
of storage (mean scores of total quality 3.1 to 3.5). After that, the sensory quality of the
pasteurised samples began to decrease in comparison to the control sample. The quality
decrease was most clearly detectable in the freshness of odour and taste as well as in colour
brightness. The differences between the experimental and control samples were not, however,
statistically significant. The texture remained good throughout the entire storage period. At
the end of the storage period the quality of all samples was still acceptable.
5.3.4 Prevalence of Listeria monocytogenes (III)
No L. monocytogenes were detected in inoculated (108 CFU/g) roes pasteurised for 10 min at
65 °C or at 62 °C with enrichment cultivation. The pasteurisations were stronger than
theoretical pasteurisation at the same temperatures and duration due to the exclusion of the
heating up and cooling down times in the theoretical calculations. The roe jars were at
elevated temperatures (> 50 °C) for 56 min and 51 min at 65 °C and at 62 °C pasteurisations,
respectively, which are five times longer than the actual pasteurisation time of 10 minutes.
5.3.5 Pasteurisation values (III)
In order to assess the effectiveness of the different roe pasteurisation treatments,
pasteurisation values were calculated for experimental pasteurisations and for theoretical 1D
pasteurisation based on the determined D-values at 60 °C and 63 °C. The effect of the heating
up and cooling down processes is included in the pasteurisation values of the experimental
pasteurisations. Pasteurisation values ranged between 73 to 247 minutes and the
corresponding 1D treatment pasteurisation value was 1.6 min. Based on the division of these
figures the performed experimental pasteurisations at 62 °C and 65 °C destroyed 46 to 154
log units of L. monocytogenes cells. In other words, the mildest pasteurisation temperature
(62 °C, 10 min) corresponds to almost four times the theoretical 12D treatment.
40
5.4 Occurrence of Listeria monocytogenes and Listeria spp. and surface hygiene in fish
processing factories (IV)
L. monocytogenes and Listeria spp. were found from 30% and 65% (7/23 and 15/23) of the
factories sampled, respectively. In the factories that were Listeria spp. positive, 7.2% of all the
surfaces sampled tested positive. One-third of the positive samples identified were
L. monocytogenes and the rest were mainly L. innocua. Listeria spp. was found from e.g. the
brine basin, brine machine, packaging table, cold-smoke skewer, grindstone, skinning
machine, vacuum packaging machine, scale, apron, conveyor belt, door handle, light switch,
grilling machine, floors and drains.
About half (48%) of the washed and cleansed surfaces of the fish factories were at least
moderately contaminated with aerobic bacteria (> 1.8 CFU/cm²) and one-fourth (26%) of all
the samples were heavily contaminated (> 5 CFU/cm²). In 43% of the samples ATP activity
exceeded 1 RLU that indicates evident microbial activity. The amount of enterobacteria was
over 0.1 CFU/cm² in 20% of the samples and in 9% of the samples the amount of
enterobacteria exceeded 1.0 CFU/cm². One-third of the surfaces were at least moderately
(> 0.6 CFU/cm²) contaminated with moulds and over 10% of the surface samples exceeded
1.6 CFU/cm². Yeast contamination was only slightly lesser than the mould contamination. Of
the Listeria spp. positive surfaces, 71% (22/31) were at least moderately (> 1.8 CFU/cm²)
contaminated, and 68% were heavily contaminated (≥ 5 CFU/cm²) in terms of total bacteria
counts, and 59% had an ATP result higher than 1 RLU. Enterobacteria (≥ 0.1 CFU/cm²) were
found in 56% of the Listeria spp. positive samples. Yeast and mould contaminations of the
Listeria spp. positive surfaces were 44% and 43%, respectively. On the other hand, 8.8% and
7.5% of the all enterobacteria and aerobic bacteria contaminated surfaces were also
Listeria spp. contaminated.
Almost half (45%) of the ready-to-eat fish products had Listeria spp. contamination.
L. monocytogenes was found in 13% of the ready-to-eat fish products, including cold-smoked
(17%) and hot-smoked fish (8%), cold-salted fish (20%) and roe (0%). The contamination of
raw fish was slightly lesser (33%) for Listeria spp. and (11%) for L. monocytogenes.
Listeria spp. was detected from the raw fish or fish products in 70% (16/23) and
L. monocytogenes from 30% (7/23) of the factories. In five factories all product samples
examined had Listeria spp. contamination and one out of three brines examined had
L. monocytogenes contamination. In this factory L. monocytogenes contamination occurred in all
the products studied.
41
5.5 Contamination sources of Listeria monocytogenes at the fish farm (I, V)
During the three year survey, 17% (41/240) of the samples taken from the farm and its
surroundings were contaminated with L. monocytogenes. At the beginning of the follow-up the
surfaces, water and fish in the farming area were heavily contaminated with both
L. monocytogenes and L. innocua. All wet surfaces had Listeria spp. contamination. The
washed fish cleaning house, however, was free from Listeria spp. except for one L. innocua
contaminated drain (1/17). There was no Listeria spp. found in the ten fish collected
simultaneously from further out to sea. The contamination of the fish farm environment and
fish with L. monocytogenes varied from heavy contamination at the beginning of the follow-up
through sporadic incidence to a situation with no Listeria spp. found until a new extensive
contamination at end of the follow-up. Fig. 1 demonstrates the association of the mean
monthly rainfall and the L. monocytogenes occurrence in the fish farm. L. monocytogenes was
found in some of the samples in all sampling occasions when the mean monthly rainfall
exceeded 50 mm. No L. monocytogenes but other Listeria spp. were found in sampling
occasions when the mean monthly rainfall was less than 50 mm. In two occasion, however,
when the mean rainfall was less than 10 mm, no Listeria spp. were found in any of the
samples.
180
2001 to 2004
160
Mean 1970 to 2000
Rainfall (mm)
140
120
100
80
60
40
20
0
6 7 8
9 10 11 12 1 2
2001
3 4 5
6 7 8
9 10 11 12 1 2
3 4 5 6
2002
7 8 9 10 11 12 1 2 3
2003
4 5 6
7 8 9 10 11 12
2004
Year, month
Figure 1. The rainfall per month measured near the fish farm location from June 2001 until the end of
2004 compared with the mean rainfall during 1970 to 2000. The performed samplings are marked with
bigger symbols (■ L. monocytogenes found in sampling, ● other Listeria spp. but no
L. monocytogenes found, ▲ no Listeria spp. found). L. monocytogenes findings are clarified in
Table 11.
42
L. monocytogenes isolates (98) from the fish farm studied fell into 12 PFGE pulsotypes
(Table 11). During the follow-up time, the occurrence of different pulsotypes changed. Six
different pulsotypes were present in the fish farm at the beginning of the study, in the
following years the old pulsotypes disappeared and new ones emerged. The fish were
contaminated only two times and both times only one L. monocytogenes pulsotype was found.
These pulsotypes were not found in any other sample types. The fish contaminants belonged to
different clusters. Ribotyping of the 28 L. monocytogenes isolates from the first contamination
wave formed seven ribotypes. The same isolates formed ribotypes according to their
pulsotypes except ribotyping separated one pulsotype into two groups (Table 11).
Table 11. Dendogram, sample places, pulsotypes, ribotypes, serotypes, sampling dates and the number
of PFGE isolates (98) of L. monocytogenes isolates from one fish farm typed with pulsed field gel
electrophoresis using AscI enzyme and ribotyped with EcoRI enzyme.
Dice (Opt:1.00%) (Tol 1.0%-1.0%)
(H>0.0% S>0.0%) [0.0%-100.0%]
50
55 60 65 70 75 80 85 90 95 100
Sample
place
Pulsotype
Ribotype
Sampling
date
(month/year)
No. of
PFGE
isolates
Ground
a1
9/04
3
River water
a2
11/03
4
Brook water
b
9/04
2
Fish
Fish
c1
c1
1
10/01
1/02
19
9
Surface
Sea water
c2
c2
2
2
11/01
11/01
1
1
Fish
d
9/04
6
Sea water
Ground
e
e
11/01
5/03
1
5
Ground
f1
5/03
5
Sea water
Ground
f2
f2
11/01
6/02
2
5
Brook water
Sea water
g1
g1
9/04
9/04
1
3
Sea water and surface
Ground
Sea and brook water
g2
g2
g2
5, 6
11/01
7/02
7/03
5, 2
8
5, 5
h
7
11/01
6
Sea water
43
3
4
5.6 Distribution of Listeria monocytogenes PFGE-types isolated from different areas of
fish production chain (V)
From 15 different fish companies 181 L. monocytogenes isolates formed a total of 30 PFGEtypes. Nine of the pulsotypes comprised L. monocytogenes isolates from more than one
company (two to four companies), but most (21/30) of the pulsotypes were only found in one
company. L. monocytogenes contaminated raw fish from six fish farms harboured one or two
L. monocytogenes PFGE-pulsotypes in each fish lot. The fish processing factories and the fish
farm environment mostly harboured several L. monocytogenes pulsotypes at one sampling
time. There were nine L. monocytogenes pulsotypes found from ready-to-eat fish products.
Seven of these final product pulsotypes included isolates from other sources. Five included
isolates from a fish processing environment and four from raw fish samples, consequently two
pulsotypes included isolates from all three sources. L. monocytogenes isolates, originating
from a fish processing environment, formed 11 pulsotypes. In addition to the five pulsotypes
belonging to the final product pulsotypes, a raw fish material isolate shared one pulsotype and
five did not occur in other sample types. Raw fish isolates formed ten pulsotypes, four shared
with the final products, one with the processing environment and five not shared with any
other sample types. Ten pulsotypes found from the environment of the studied fish farm were
not shared with other sample types.
6 DISCUSSION
6.1 The role of raw fish materials as a source of Listeria monocytogenes (I, V)
The incoming raw fish material is occasionally an important L. monocytogenes source in fish
production. The L. monocytogenes prevalence in different fish lots ranged from 0 to 75% for
individual thawed fish and from 0 to 100% according to pooled unprocessed fish samples.
The mean prevalence of L. monocytogenes in pooled rainbow trout was 15% and 9% was
found for L. monocytogenes, in the individual fish samples. The actual prevalence is
something between these figures as they either overestimate or underestimate the real
situation because all fish in pooled samples were not necessarily contaminated and freezing
destroyed L. monocytogenes bacteria in some of the individual samples. The
L. monocytogenes prevalence results found in the literature varied greatly from 0 to 88%
(Table 5) for raw fresh fish. When considering all data, however, the mean prevalence of
L. monocytogenes was 14%, which is near the results in this study.
An essential factor in the transmission of L. monocytogenes contamination between raw fish
and fish processing is the location of L. monocytogenes in fish. The location of
44
L. monocytogenes in different parts of raw fish material differed significantly (p < 0.0001) in
rainbow trout. Up to 96% of the L. monocytogenes positive samples were gill samples. Only
4% (2/45) of the L. monocytogenes positive samples were skin or viscera samples. Rainbow
trout is therefore contaminated by L. monocytogenes almost exclusively in the gills and only
sporadically in the skin and viscera. On the basis of these results special effort should be
focused on the isolation and removal of rainbow trout gills before the L. monocytogenes
contamination can spread further.
The prevalence of L. monocytogenes varied greatly between different fish farms as mentioned
above from 0 to 100%. The results from other fish farm studies support the finding that
Listeria spp. contamination varies greatly between different farms. In a small Norwegian
salmon study, no Listeria spp. was found in gill, skin and guts of ten salmon (Ben Embarek et
al. 1997). In Switzerland (Jemmi and Keusch 1994), three studied freshwater rainbow trout
fish farms showed no Listeria spp. in fish at one first farm, but the other two farms had
Listeria spp. in fish skin (6/15 and 9/15) and in faecal content (6/15 and 4/15) sampled with
swabs. Gills were not studied in that investigation (Jemmi and Keusch 1994). In USA,
L. monocytogenes was isolated from combined skin and gut samples of aquacultured hybrid
striped bass in two out of three fresh water ponds (Nedoluha and Westhoff 1993). If fish grow
in areas where the presence of L. monocytogenes is possible, like polluted waters and waters
with a high content of organic material (Ben Embarek 1994), it is probable that occasionally
the fish will also harbour L. monocytogenes.
Typing of 181 L. monocytogenes isolates from 15 fish farming and fish processing factories,
including raw fish and final fish products, showed a wide range of different L. monocytogenes
pulsotypes (30). Both the raw fish and the processing environment L. monocytogenes isolates
were often found together with product isolates within the same PFGE-type. The importance
of factory environment as the main source of L. monocytogenes contamination of final
products has previously been reported (Destro et al. 1996, Autio et al. 1999, Fonnesbech
Vogel et al. 2001). This study, however, also emphasises the importance of the raw materials
as a source of L. monocytogenes at least occasionally when they are heavily contaminated.
Few other studies exist that also suggest that the raw materials have a relevant role in
contamination of products and the process environment (Eklund et al. 1995, Fonnesbech
Vogel et al. 2001, Norton et al. 2001, Markkula et al. 2005).
45
6.2 Safety and quality of Finnish roe at retail level (II)
Roe products are a potential source of L. monocytogenes in Finland. The presence of
L. monocytogenes in different fish species roe varied from 2 to 8%. Fresh-bought roes,
however, contained 20 times more often L. monocytogenes (18%) than frozen or frozenthawed roe products. This may be caused by the freezing process destroying and injuring
L. monocytogenes cells despite the fact that they are quite resistant to the freezing process and
normally only a small amount is inhibited during the frozen storage (Golden et al. 1988,
Harrison et al. 1991). In Japan, the prevalence of L. monocytogenes in roe products in retail
markets was 10% (Handa et al. 2005) which is quite near the values found here. The detected
amounts of L. monocytogenes were low (< 100 CFU/g), which supports the inactivation effect
of freezing. The storage time of fresh or frozen-thawed roe, before consumption, is only a few
days and therefore the risk of the development of high counts of L. monocytogenes is
relatively low if the roe is kept appropriately refrigerated (< 3 °C).
Wide variations in the microbial as well as sensory quality of the roe products exist. The
microbial quality was poor in 57% of the roe samples, having aerobic bacteria over
107 CFU/g, and 73% of the samples had coliforms over 150 CFU/g. The overall microbial
quality was worst in vendace roe. This is probably because the small size of the ovaries
necessitates more handling during production. Jokinen et al. (1992) also found high bacterial
counts (107-8 CFU/g) in 62% of salmon, rainbow trout, whitefish and vendace roes bought
from wholesale and retail markets in Sweden. They found, however, only small amounts of
coliform bacteria. Himelbloom and Crapo (1998) found that aerobic counts increased in pink
salmon ikura as the 30-day Alaska production season progressed, ending with 4.5 x 107 CFU/g
in fresh eggs. The total coliform counts during the season varied from below a detectable
level to 2.4 x 103 CFU/g. These results are similar to the findings in the present study.
The correlations between microbial and sensory measures were low. Unacceptable quality in
the taste of freshness, by sensory analyses, was found in 20% of the roe samples. Roe samples
bought fresh had a better sensory quality than products bought frozen or frozen-thawed. The
deterioration in quality was often reflected in all evaluated sensory characteristics, partly
because olfactory and taste perceptions as well as chemical feeling factors are all perceived in
the mouth and the flavour terms easily intermingle (Meilgaard et al. 1999). Defects were most
consistently found in vendace roe samples and the rainbow trout roe was rated as the freshest.
Musty and rancid off-odours and off-tastes were detected in roes from every fish species.
Yeast-like or mould-like off-odours and off-tastes were also perceived in many samples.
46
These defects suggest the presence of temperature abuse along the cold chain or poor
production plant hygiene. Rancidity is a common indicator of time temperature-dependent
spoilage of fish material (Huss 1995). Too long of a storage time can also explain
deterioration in quality, especially in frozen-thawed roe, since there was often no best before
expiration date on the products sold at service counters.
6.3 Safety and quality of pasteurised rainbow trout roe (III)
Pasteurisations of rainbow trout roe for 10 min at 62 °C and 65 °C were effective in
eliminating inoculated L. monocytogenes cells (108 CFU/g), which is about the highest
possible L. monocytogenes cell count to grow in rainbow trout roe. Experimentally
determined D- and z-values for the mixture of four L. monocytogenes strains were a little
lower than the values found in the literature on fish or crab meat (Ben Embarek and Huss
1993, Mazzotta 2001, Table 4). Based on the determined D- and z-values, however, the
experimental pasteurisations were calculated to eliminate at least 45 log units of
L. monocytogenes that is sufficiently even in case of a more heat resistant L. monocytogenes
strain.
The quality of cold stored pasteurised vacuum packaged rainbow trout roe, stored for six
months, was consistently good in terms of microbial as well as sensory quality.
Pasteurisations inactivated most of the bacteria in rainbow trout roe. The initial bacterial load
in roe, however, was relatively low. In addition, B. circulans was identified from roe
pasteurised for 10 min at 62 °C, which demonstrates that bacterial spores can survive
pasteurisation. The storage temperature of the pasteurised roe has to be below 3 °C to prevent
spore-forming bacteria from multiplying and forming toxins, especially since there is a risk of
group II C. botulinum spores being found in the fish roe (Hyytiä et al. 1998, Lindström et al.
2004). In addition, the low storage temperature is important to ensure the stability of the
sensory quality of roe. Inhibition of lipid hydrolysis is the main way to ensure sensory quality
of roe products stored for an extended time period. Enzymatic lipid hydrolysis is effectively
retarded by either freezing or pasteurisation (Kaitaranta 1982). In addition, vacuum packaging
is essential in minimizing the oxidative rancidity of lipids. The lipid hydrolysis was
successfully avoided as the sensory quality of all the roe samples remained good after a six
month storage period. Pasteurisation offers a feasible choice for the production of safe, good
quality rainbow trout roe products for consumers.
47
6.4 Listeria monocytogenes, Listeria spp. and surface hygiene in fish processing factories
(IV)
L. monocytogenes and Listeria spp. were found on surfaces of one-third and two-thirds of the
factories, at least sporadically. Many important process surfaces were contaminated with
Listeria spp. The washing and cleaning practices in most (26/28) of the studied fish factories
were inadequate and the cleaned process surfaces were contaminated with aerobic bacteria,
enterobacteria and fungi. The Listeria spp. positive surfaces contained increased (> 1.8
CFU/cm²) numbers of aerobic bacteria in 71% of the samples. In comparison to another fish
industry study in which 94% of the Listeria spp. positive surface samples were contaminated
with aerobic bacteria (Aarnisalo et al. 1998). Surface samples having increased amount of
enterobacteria or aerobic bacteria, were two and one and a half times more likely
contaminated with Listeria spp. than samples with increased ATP levels or fungi amount.
None of the studied hygiene monitoring methods or microbes was an effective indicator of the
presence of Listeria spp. on the surfaces. The presence of Listeria spp. on the factory surfaces
was, however, an indicator of its increased possibility to be found in the fish products. In
factories where Listeria spp. was found on surfaces it was often found (10/13) in some
products too. The contamination of different ready-to-eat fish products with L. monocytogenes
was the same level (0 to 20%) as previously found in Finland (Johansson et al. 1999).
The contact plate surface detection method for Listeria spp. was easy to perform and
preliminary results were obtained rapidly. On the other hand, the effectiveness and reliability
of the contact plate method was low as the sampled area was small (25.5 cm²) and the stressed
Listeria spp. cells from the washed surfaces may have suffered from the selective compounds
of the agar. Disruptive microbial populations, due to the lack of enrichment stages, also
complicated the identification of Listeria spp. colonies.
6.5 Sources of Listeria monocytogenes in fish farming (I, V)
During the three year follow-up period, the primary routes of L. monocytogenes
contamination of the fish farm were clarified. The fish were contaminated with
L. monocytogenes in the costal farm area, as the fish farmed further out to sea were Listeriafree, but became heavily contaminated in the costal farm area. The thorough contamination of
the fish lot occurred within a four-week period when the fish lot was kept in the coastal area
before sampling. Interestingly, the typing results revealed that the two L. monocytogenes
PFGE-types found in the fish samples during the follow-up were different from the
L. monocytogenes PFGE-types isolated from the water environment at the fish farm. The
48
brook and river waters, as well as other runoff waters, seemed to be the main contamination
source at the fish farm, regardless of the fact that the typing did not reveal any place to be the
main L. monocytogenes source. Twice the same L. monocytogenes PFGE-type was found in
the brook and sea waters (Table 11) at the same time, and once L. monocytogenes was found
in the river water, but not in the nearby sea water. Listeria spp. was also found in the river and
brook waters, but not in sea water samples taken at the same time. The results by Colburn et
al. (1990) support this conclusion, as they found that the freshwater tributaries draining into
Humboldt-Arcata Bay were consistent sources of Listeria spp. The primary L. monocytogenes
source contaminating the river and brook waters was not extensively studied. There was no
specific source, like agriculture or cattle, that could be suspected of direct contamination of
water. On the other hand, it is possible that L. monocytogenes may naturally be present, at
least sometimes, in the soil of a river or brook, as it is frequently found in soil and water
environments (Weis and Seeliger 1975, Ben Embarek 1994, Schaffter and Parriaux 2002).
The widespread presence of L. monocytogenes in soil has often been attributed to
contamination from decaying plant and faecal material, with damp surface soil providing a
cool, moist protective environment and the decaying material the substrate, which together
enable the survival of L. monocytogenes from season to season (Fenlon 1999). This may be
the case, to some degree, with L. monocytogenes in wet soil environments like river, brook
and sea bottom soils.
Weather conditions had a strong influence on the probability of finding L. monocytogenes and
other Listeria spp. in the fish farm environment (Fig. 1). The number of samples contaminated
with Listeria spp. was typically larger after rainy periods. The occurrence of
L. monocytogenes and Listeria spp. in samples decreased, however, during dry periods. In
addition, it was also noted that when sampling was performed immediately after a few days of
rain showers during a relatively dry period the same L. monocytogenes PFGE-type was found
in the brook and sea water. This suggests that the L. monocytogenes contamination in a water
environment is a phenomena occurring rapidly if a suitable source is present in the
environment and the weather conditions are favourable, but disappearing soon if no further
contamination arrives.
The original L. monocytogenes contamination in fish gradually disappeared. The time needed
for this was several months. Such disappearance was faster in sea water than in fish. Hsu et al.
(2005) showed that L. monocytogenes does not readily survive in sea water and does not
compete well with microbial marine population at elevated temperatures (22 °C) in the
49
presence of organic material. At temperatures below 11 °C, L. monocytogenes lost viability
throughout storage but was detectable after six days of incubation (Hsu et al. 2005). The soil
samples often contained L. monocytogenes PFGE-types found earlier in other sample types,
even 18 months after the first discovery. It is possible that the L. monocytogenes strains did
not survive in the soil for the entire period, but reappeared via e.g. brook water. It has,
however, been shown earlier that many bacteria including L. monocytogenes survive longer in
sediments than in water (Botzler et al. 1974, Burton et al 1987). The difference in the
occurrence of Listeria spp. in different sea bottom soil samples was clear. No Listeria spp.
was found from the deeper sea areas whereas all inshore samples taken at the same time
contained L. monocytogenes or other Listeria spp.
It has been established that cattle contribute to the amplification and dispersal of
L. monocytogenes in a farm environment and that ruminant farm ecosystems maintain a high
prevalence of L. monocytogenes (Nightingale et al. 2004). This fish farm did not seem to be
contributing to the maintenance of the amplification and dispersal of L. monocytogenes in the
water environment. On the contrary, the fish did not become contaminated easily and only
two out of twelve L. monocytogenes strains found from the farm were also found in fish. In
addition, the viscera of rainbow trout did not contain L. monocytogenes at this fish farm and
overall the viscera were contaminated only once with L. monocytogenes in all the studied
rainbow trout samples (Table 10). After gavage ingestion of live L. monocytogenes cells by
salmon no L. monocytogenes were detected after three and seven days in the fish stomachs
(Hsu et al. 2005). Thus, fish do not spread and increase the environmental contamination with
L. monocytogenes contaminated faeces, since the bacterium is not persistent in fish. The fish
farm studied did not spread Listeria contamination, but on the contrary suffered from
L. monocytogenes contamination from outside sources like the brook water.
50
7 CONCLUSIONS
1.
The prevalence of L. monocytogenes in pooled unprocessed fresh rainbow trout was on
average 15%. In the individual thawed fish samples were found a prevalence of 9% for
L. monocytogenes. The prevalence of L. monocytogenes varied greatly between different
fish farms: from 0 to 100% in pooled samples and from 0 to 75% in individual fish
samples. The location of L. monocytogenes in different parts of the raw fish material
differed significantly (p < 0.0001) in rainbow trout. Rainbow trout contaminates with
L. monocytogenes almost exclusively in the gills and only sporadically in the skin and
viscera. Up to 96% of the L. monocytogenes positive samples were gill samples and only
4% of the L. monocytogenes positive samples were skin or viscera samples. Special effort
should be focused on the isolation and removal of the gills of rainbow trouts before the
L. monocytogenes contamination spreads further.
2.
The presence of L. monocytogenes in different Finnish fish species roe products in the
retail market varied from 2 to 8%. Especially fresh-bought roe products contained
significantly (p < 0.01) more often L. monocytogenes (18%) than frozen and frozenthawed roe products (0.9%). The microbial quality of the roe samples was poor in
relation to aerobic and coliform bacteria in 57% and 73% of the samples, respectively,
plus 20% of the roe samples were unacceptable to taste.
3.
Based on the determined D- and z-values for four L. monocytogenes strain mixture the
performed pasteurisations at 62 °C and at 65 °C for 10 min theoretically destroyed 46 to
154 log units of L. monocytogenes cells in rainbow trout roe. The experimental
pasteurisations of L. monocytogenes inoculated rainbow trout roe samples, carried out at
62 °C and at 65 °C for 10 min, destroyed 8 log units of L. monocytogenes, which has
shown to be the highest possible L. monocytogenes cell count to grow in roe. The
experiments performed showed that the quality of pasteurised vacuum packaged rainbow
trout roe was consistently good regards to of both microbial and sensory quality for up to
6 months when stored at 3 °C. Pasteurisation offers a feasible choice for the production of
safe, good quality rainbow trout roe products for consumers. The storage temperature of
the pasteurised roe, however, has to be below 3 °C to prevent the formation of toxins,
especially as there is a risk for the growth of group II C. botulinum spores.
51
4.
L. monocytogenes and Listeria spp. were found on surfaces of one-third and two-thirds of
the fish factories at least sporadically. The surface contact agar and ATP-based
techniques were not effective indicators of the presence of Listeria spp. on the surfaces.
The presence of Listeria spp. on the factory surfaces was, however, an indicator of its
increased possibility to be found in the fish products. In factories where Listeria spp. was
found on surfaces it was often (10/13) found in some products. L. monocytogenes
contamination level of different ready-to-eat fish products varied from 0 to 20%.
5.
The brook and river waters, as well as other runoff waters from environment, were the
main contamination sources of L. monocytogenes in the studied fish farm. Weather
conditions had a strong influence on the probability of finding L. monocytogenes and
Listeria spp. in the fish farm environment and in fish. The number of samples
contaminated with L. monocytogenes and Listeria spp. was often greater after rainy
periods. The occurrence of L. monocytogenes and Listeria spp. in samples decreased
during dry periods. The L. monocytogenes contamination in fish gradually disappeared
over several months. Such disappearance, however, was faster in the surrounding sea
water than in the fish. Presence of certain L. monocytogenes PFGE-types, after the first
discovery months earlier in some other sample type, was typical for sea bottom soil
samples. The two L. monocytogenes PFGE-types found in fish were different than the
L. monocytogenes PFGE-types found elsewhere in the fish farm environment. The fish
farm studied did not spread L. monocytogenes contamination, but on the contrary suffered
from L. monocytogenes contamination from environmental sources.
6.
L. monocytogenes PFGE-types, isolated from raw fish and from fish production
environments, were found also among final fish products. Raw fish materials and
production environment and machines are sources of L. monocytogenes contamination
that both need to be monitored.
52
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